CN101500597A - Receptor antagonists for treatment of metastatic bone cancer - Google Patents

Abstract

The invention provides methods of treating bone cancer, particularly metastatic bone cancer, by administering an IGF-IR antagonist and/or a PDGFRa antagonist. The invention also provides antibodies that bind to human PDGFRa and neutralize activation of the receptor. The invention further provides a method for neutralizing activation of PDGFRa, and a method of treating a mammal with a neoplastic disease using the antibodie alone or in combination with other agents.

Description

The receptor antagonist of treatment metastatic bone cancer

[0001] rights and interests of No. the 60/691st, 192, the claimed U.S. Provisional Application of the application.

Invention field

[0002] the invention provides by giving the especially method of metastatic bone cancer of IGF-IR antagonist and/or PDGFR alpha-2 antagonists treatment osteocarcinoma.The present invention also provide combine with people PDGFR α and in and the antibody of the activation of this receptor.In having the present invention further provides and the method for the activation of PDGFR α and use this antibody separately or unite the method for using the treatment tumor disease with other drug.

Background of invention

[0003] carcinoma of prostate is the most general cancer of male, and the U.S. has 220,000 routine carcinoma of prostate every year approximately, dead 29000 examples.Diagnosed the male who suffers from carcinoma of prostate all to suffer from the cancerometastasis disease greatly.And although accepted operation or radiotherapy, cancer metastasis finally still can take place in many patients with prostate cancer.Bone is the modal transfer of carcinoma of prostate site, also is the common transfer site of breast carcinoma and pulmonary carcinoma simultaneously.It is androgen dependent form that many carcinoma of prostate shift, so produced many Therapeutic Method of castrating rapidly by operation or medicine, but in nearly all case, this cancer can finally become non-androgen dependent form, causes very high M ﹠ M.In case the bone transfer has taken place, the effect of existing therapy is very limited.So far the therapy (giving docetaxel) of Bao Dao the most effective and approved treatment metastatic prostate cancer can prolong median survival about three months.(Petrylak etc., 2004, N.Engl.J.Med.351:1513; Tannock etc., 2004, N.Engl.J.Med.351:1502).Therefore, we are badly in need for the treatment of the method for metastatic bone cancer at present.

[0004] IGF-1 (IGF-IR) is a kind of ubiquitous film tyrosine kinase receptor of striding, and is essential to normal period of fetus and birth growth of later stage with growing.IGF-IR is positioned at the surface of most cell types, as the signal transducers of growth factor I GF-I and IGF-II (below be referred to as IGFs).IGF-IR can stimulate cellular proliferation, the change of cell differentiation, cell size and stop apoptosis.It is considered to that pair cell transforms is to be close to essential (row summary Adams et al. as follows, Cell.MoI.Life Sd.57:1050-93 (2000); Baserga, Oncogene 19:5574-81 (2000)).There is report to show the expression height of IGF-IR in the tissue sample of carcinoma of prostate metastatic tumor of bone.The storage capacity of IGFs is that human body is maximum in the bone.

[0005] IGF-IR is a kind of preformed allos tetramer, comprises by disulfide bond covalently bound two α chains and two β chains.The subunit of this receptor is synthesized as the part of a 180kd single polypeptide chain, and this polypeptide chain generates α (130kd) and β (95kd) subunit through Proteolytic enzyme processing back again.Whole α chain all is positioned at the extracellular and comprises and the bonded site of part.The β chain has membrane-spanning domain, tyrosine kinase domain and terminal extension of C-, and this end extension is essential for cell differentiation and conversion, but for mitogen signal transduction and prevention apoptosis and nonessential.

[0006] IGF-IR and Insulin receptor INSR (IR) have high similarity, especially the sequence of β chain (70% homology).Just because of this homology, recent many researchs show all that these receptors can form and contain an IR dimer and a dimeric heterozygote of IGF-IR (Pandini et al., Clin.Cane.Res.5:1935-19 (1999)).In normal cell and transformant, all can form this heterozygote, and the content of this heterozygote depends on the concentration of two kinds of homodimer receptors (IR and IGF-IR) in the cell.Though the heterozygosis receptor is made of in pairs IR and IGF-IR, but this heterozygote combines with IGFs specifically, and affinity is similar to IGF-IR's, with weak (the Siddle and Soos of combining of insulin, The IGF System.Humana Press, pp.199-225.1999).Therefore these heterozygotes can be incorporated in conducted signal in normal cell and the transformant with the IGFs knot.

[0007] second kind of IGF receptor IGF-IIR or be called Man-6-P (M6P) receptor, also can combine with the IGF-II part with high-affinity, but but there is not tyrosine kinase activity (Oates et al., Breast Cancer Res.Treat.47:269-81 (1998)).This is because it can cause the degraded of IGF-II, is considered to the meeting point of IGF-II, growth promoting function that can this part of antagonism.The minimizing of IGF-IIR can it promotes growth to IGF-II and the bonded antagonism of IGF-IR by performance in the tumor cell.(Byrd?et?al.，J.Biol.Chem.274：24408-16(1999)).

[0008] deriving from hematoblastic α and β growth factor receptors (PDGFR α and PDGFR β) is III-receptor tyrosine kinase.PDGFR α brings into play pivotal role and also brings into play important function in the adult phase in growth.For example, the mice homozygote of null mutation can death when the embryo forms.Express in many intercellular substance structures at development later stage PDGFR α, and adjacent epithelial cell produces the somatomedin (PDGF) in platelet source.All detect α in the normal or prostatic tissue sample of hypertrophy less than PDGFR, yet, PDGF-B expression R α all in contrast experimenter's former prostate cancer tissue and the sclerotin.And from the prostate cell line that different cancerometastasis site obtains, bone shifts the PC3 cellular expression PDGFR α in source, but does not express from the cell line of lymph node (LNCaP) and the acquisition of brain (DU-145) tissue metastasis.

[0009] growth factor family in the platelet of somatomedin source is made up of the dimer of five kinds of different disulfide bondings, PDGF-AA ,-BB ,-AB ,-CC and-DD, it plays a role by PDGFR α and PDGFR β.These somatomedin are chimeric molecules, by can be simultaneously in conjunction with the disulfide bonding of two receptor proteins and induce the polypeptide chain of receptor dimerizationization, autophosphorylation and intracellular signal transduction to form.Similar on PDGFR α and the PDGFR beta structure, and can form heterodimer and homodimer.Because the binding affinity of PDGFR β and PDGF-A chain is not strong, PDGF-AA only activates α α receptor dimer, and PDGF-AB and PDGF-CC then activate α α and α beta receptor heterodimer.

Summary of the invention

[0010] the present invention relates to treatment, comprise the tumor that is derived from prostate, mammary gland or lung and expression of insulin like growth factor-I receptor (IGF-IR) and/or derives from hematoblastic α growth factor receptors (PDGFR α) constitutional and metastatic tumor of bone.

[0011] tumor of being treated can be hormone/androgen dependent form or hormone/androgen independent form, and can be to be derived from prostate, the tumor of mammary gland or lung.

[0012] the invention provides treatment bone tumor patient's method and the method that suppresses the bone tumor growth.Described method comprises the IGF-IR antagonist that gives effective dose or the PDGFR alpha-2 antagonists of effective dose.Described receptor antagonist comprises micromolecular inhibitor in antibody and antibody fragment and the cell.

[0013] the invention provides and with its target receptors bind and to suppress the bonded anti-IGF-IR of part or anti--PDGFR Alpha antibodies.The present invention also provide can in and activated antibody and other antagonisies of IGF-IR or PDGFR α.Some antibody can be reduced their target receptor by for example internalization and/or Degradation in addition.Therefore the effect of described antibody and micromolecule antagonist is to suppress for example activation of Akt, p42/p44 and MAPK of downstream signal transduction molecule.

[0014] described method comprises independent use IGF-IR or PDGFR alpha-2 antagonists, and the two uses simultaneously or for example use is united in chemotherapeutics and radiotherapy with other cancer therapies.

[0015] the present invention also provide can with PDGFR α and nucleotide bonded antibody and antibody fragment and the host cell that is used to produce antibody.Described antibody can block ligand combination and in and the activation of receptor.The present invention also provides the described antibody of independent use, has united the treatment of using or being used for tumor disease as conjugate with other receptor antagonists or antitumor drug.Anti--PDGFR Alpha antibodies can be used for many tumor treatment, for example: ovarian tumor, breast tumor, pulmonary carcinoma, hepatocarcinoma, gastrointestinal stromal tumor, melanoma, renal cell carcinoma, tumor of prostate and soft tissue sarcoma.

Description of drawings

[0016] Fig. 1 has described and castrated SCID mice LuCaP35V subcutaneous transplantation growth of tumor in a treatment stage, and this treatment reaches 150-200mm from tumor ³In time, begin to carry out.A group: the matched group of not receiving treatment; B group: use docetaxel (10mg/kg or 20mg/kg) separately or unite use animal is scheduled to last treatment all around with anti-IGF-IR antibodies (40mg/kg IMC-A12); C group: the mice serum PSA level of the load LuCaP 35V subcutaneous transplantation tumor of not receiving treatment and receiving treatment.Accepted by the mice treated that docetaxel (20mg/kg) is treated separately or the therapeutic alliance of docetaxel (10mg/kg or 20mg/kg) and anti-IGF-IR antibodies (40mg/kg IMC-A12).Treatment from tumor growth to 150-200mm ³The time, finish the back all around.

[0017] Fig. 2 represent that docetaxel (10mg/kg) (A group) is treated separately or with anti-IGF-IR antibodies (40mg/kg IMC-A12) (B group) therapeutic alliance after the single cell suspension of LuCaP 35V transplantation tumor.The region representation of mark R1 contains the apoptotic cell (rising of FITC labelling) of sheet segment DNA.

[0018] Fig. 3 represents that docetaxel (10mg/kg or 20mg/kg) is treated separately or finishes the DNA synthetic (BrDu absorption) of transplantation tumor afterwards with anti-IGF-IR antibodies (40mg/kg IMC-A12) therapeutic alliance.

[0019] Fig. 4 has described with after docetaxel and A12 therapeutic alliance and the independent treatment of docetaxel, with tumor of prostate invasion and attack (TUBB), the differential expression of antiandrogen therapy resistance (BIRC5) and apoptosis-inducing related gene.

[0020] Fig. 5 represents to treat the serum levels that stops back A12.

[0021] Fig. 6 animal via of representing not suffer from disease cross that successive docetaxel (10mg/kg or 20mg/kg) is treated separately or with anti-IGF-IR antibodies (40mg/kg IMC-A12) therapeutic alliance after body weight (a kind of total Cytotoxic measuring method).

[0022] Fig. 7 represents the therapeutical effect of anti-IGF-IR antibodies (IMC-A12) to the PSA of graft generation in the SCID mice of transplanting LuCaP 23.1 cells.

[0023] Fig. 8 represents to transplant a series of X skiagrams of the SCID mice of LuCaP 23.1 cells.A12 mouse peritoneal injection 40mg/ml IMC-A12, inferior on every Wendesdays, continued for six weeks.Carrying out the X line when putting to death animal takes pictures.

[0024] Fig. 9 represents PSA level (a) of the SCID mice of LuCaP 23.1 human prostate cell transplantation tumors in the load tibia and representative radiograph (b).

[0025] Figure 10 has described people's bone marrow aspiration liquid to the active effect of prostate gland cancer cell Akt.Cell lysate is carried out sds polyacrylamide gel electrophoresis.In Western blot analyzes, be phosphoric acid-Akt (Ser-473, cell signalling technology) with the thin film target, PDGFR α (R﹠amp; D Sysstems) and the antibody trace of actin (Sigma).Use with bonded protein A of HRP or Protein G (Sigma) and detect anti-combining.

[0026] Figure 11 has described inducing of AKT-phosphorylation in the PC3-ML cell and has suppressed.AG-1296 was to the dose dependent inhibitory action of Akt phosphorylation under A group expression 30ng/ml PDFG-BB existed.The inhibitory action of B group expression bone marrow aspiration liquid Akt phosphorylation and 20 μ M AG-1296.The expression of C group is compared the effectiveness that bone marrow aspiration liquid is induced the Akt phosphorylation with 100pg/ml PDGF-AA with 100pg/ml PDGF-BB combined induction effectiveness.D group has compared bone marrow aspiration liquid and has induced Akt phosphorylation, the AG-1296 size to the Akt phosphorylation of the inhibition of the inductive Akt phosphorylation of bone marrow aspiration liquid and PDFG-AA+PDFG-BB combined induction.

[0027] Figure 12 has described the inhibitory action of PDGFR alpha-2 antagonists to PC3 cell Akt phosphorylation.IMC-3G3 is to the dose dependent effect by the inductive Akt phosphorylation of 30ng/ml PDGF-BB for A group expression monoclonal antibody.B and C group has compared IMC-3G3 and the AG1296 effect to the inductive Akt phosphorylation of bone marrow aspiration liquid.The D group has shown that the inhibition to the Akt phosphorylation depends on the precincubation time of IMC-3G3.

[0028] Figure 13 has shown combining of antibody and PDGFR α.A: anti--PDGFR Alpha antibodies directly combines with immobilization PDGFR α extracellular domain.B:[ ¹²⁵I] PDGF-AA and the bonded inhibition of immobilization PDGFR α.

[0029] Figure 14 has shown that the specificity to PDGFR α phosphorylation and downstream effect device molecule suppresses.

[0030] Figure 15 shown mAbs to the activated PAE Ra of PDGF-AA-cell [ ³H]-inhibition that thymidine mixes.

[0031] Figure 16 shown in SKLMS-1 (A) and U118 (B) cell by the activated inhibition of the inductive downstream effect device of PDGF-AA molecule.

[0032] Figure 17 shown mAbs to the activated U118 of PDGF-AA-(A) and SKLMS-I (B) cell [ ³H] the thymidine inhibition of mixing.Also shown activated SKLMS-I of PDGF-BB (C) and U118 (D) cell [ ³H] the thymidine inhibition of mixing.

[0033] Figure 18 has shown established U118 (glioblastoma in the nude mice; A organizes) and SKLMS-I (leiomyosarcoma; B group) transplantation tumor the dose dependent therapeutical effect.

[0034] Figure 19 has shown the reduction of PDGFR α phosphorylation in the body in the U118 tumor of comparing the anti-PDGFR Alpha antibodies treatment of use with the non-specific human IgG treatment of use.

[0035] Figure 20 has described and has been used to clone the people VH that is derived from hybridoma and the GS expression vector of V κ variable region gene and The expressed people heavy chain (IgGl) and light chain protein.These two carriers be according to the method recombination to construct among the embodiment and with the recombinant vector transfection to the NSO cell.

Detailed Description Of The Invention

[0036] the present invention relates to use with insulin-like growth factor-I receptor (IGF-IR) combination Antibody or antibody fragment treatment bone tumour. The endocrine of IGF-I is expressed and is mainly transferred by growth hormone Control is also generated by liver, but organizing of other types also can be expressed IGF-I and comprise and storing The bone of the raised growth factor. According to the difference of tumor cell type, IGF-I can participate in endocrine, Paracrine and/or Autocrine regulation (Yu, H.and Rohan, J., J.Natl.Cancer Inst. 92:1472-89 (2000)).

[0037] experiment is found can be used for the antibody of IGF-IR combination treating to express IGF-IR's Bone tumour. Described antibody can use separately or unite use with other cancer therapies, especially Chemotherapeutics. Use separately the anti-IGF-IR therapy or comprise that with other one or more are antitumor The therapy of medicine (for example chemotherapy or radiotherapy) is united use and is had remarkable efficacy. To tumor growth Inhibition usually follow apoptotic increase, and may persist to all treatments and finish, however only The animal tumor that has carried out chemotherapy begins again growth.

[0038] research finds that also PDGFR α plays a significant role in the growth of bone tumour. For example, The tumor cell line of some PDGF-B expression R α preferentially is transferred to bone. Solvable with appearance in the marrow Sex factor is corresponding, and these clones demonstrate PDGFR α activation and the downstream signal transduction divides Sub-phosphorylation strengthens. Marrow is reduced by the PDGFR alpha-2 antagonists the activation of PDGFR α or is complete The full inhibition, can be by signal transduction and other acceptor junket ammonia through PDGFR α under usual condition The phosphorylation of the downstream signal transduction molecule that the acid kinase system activates also is greatly diminished. Some numbers According to the show, PDGFR α conduction not only can be by directly activating the part of PDGFR α, also Can by exist in the marrow can the trans-activation this receptor the factor activate PI3K/Akt Survival route.

[0039] primary bone tumor that can be treated according to the present invention includes but not limited to osteosarcoma, soft Osteosarcoma, fibrosarcoma and angiosarcoma. It should be noted that pernicious Secondary cases (metastatic) Tumour is more common than primary bone tumor. Passable according to the metastatic tumor of bone that the present invention can be treated Multiple source is arranged, and modal is prostate cancer, breast cancer and lung cancer. Coming of metastatic bone cancer The source can obviously be found out from medical history. Described tumour may be osteogenic also may be molten bone . Tumour may depend on the activation of IGF-IR when taking place, or transfers afterwards the IGF-IR dependence to Type. For example, being initiated with hormone/male sex hormone dependent form also can pass through male sex hormone or sharp Plain prostate cancer or the prostate cancer that generates inhibited physics or chemotherapy control Transfer may be transformed into non-hormone/male sex hormone by the sensitiveness enhancing that IGF-IR is activated and comply with Rely type. Except hormone/and the therapy of male sex hormone dependent form tumour is provided, the present invention also can answer Be used for the treatment of hormone/and male sex hormone dependent form tumour and do not need to depend on to male sex hormone or The inhibition of hormone for example can be by giving IGF-IR antibody and antineoplastic simultaneously. This A little tumours are included in the bone that activates by IGF-IR in the environment that is rich in IGF-IR in the bone and shift swollen Knurl, but they may be responsive also do not have responsive at the environment that does not contain IGF to hormonal stimulation The degree of middle growth. Not necessarily need to remove hormone for such tumour.

[0040] PDGF-dependent form bone tumour and " marrow " dependent form tumour also can be according to the present invention Treat. Soluble factor is corresponding with occurring in the marrow, can occur in the marrow dependent form tumour PDGFR α activates. For example institute is illustrational here, when a kind of metastatic and expression The human carcinoma cell line of PDGFR α with can occur that PDGFR α activates after bone marrow aspiration liquid contacts and The Akt phosphorylation. A kind of anti-PDGFR Alpha antibodies and a kind of little molecule PDGFR alpha-2 antagonists can PDGFR α activates and the Akt phosphorylation in the clone to suppress respectively. Can activate PDGFR α The solubility bone marrow factor include, but are not limited to PDGF-AA and BB.

[0041] although this marrow dependence relates to the signal transduction through PDGFR α, also has It may not merely be the combination of a kind of PDGFR alpha ligands and PDGFR α. For example lift here The example explanation, it should be noted that specific part (PDGF-AA and BB) is to PDGFR α Activation is weaker than the activation of bone marrow aspiration liquid. And, observe and find to exist when bone marrow aspiration liquid The time, along with the prolongation Akt phosphorylation reduction of incubative time. Considering these results shows and removes To outside reacting with the combination of FDGFs, PDGFR α also may be by to other marrow Other Signal Transduction Components of composition sensitivity (for example other receptor tyrosine kinases) trans-activation (phosphorylation). In either case, in the clone (example that is suitable for being transferred to the bone growth As, preferentially be transferred to Bone cells system) in can observe marrow dependent form PDGFR α Activation, this activation can be suppressed by the PDGFR alpha-2 antagonists. And, use PDGFR α short of money Anti-dose treat the activation that can suppress the anti-apoptosis pathway of PI3K/Akt that marrow induces and by Mitogen-activated protein kinase (MAPK).

[0042] using the bone tumor of PDGFR alpha-2 antagonists treatment can be to shift from prostate gland cancer cell, and may be hormone/androgen dependent form as mentioned above or be transformed into hormone/androgen independent form.These tumors also can shift from non-prostate gland cancer cell.Those skilled in the art can use known traditional detection method that these states and disease are made diagnosis at an easy rate.

[0043] treatment means and comprises any treatment to a certain disease of animal: but (1) is being infected certain disease is not easily also being suffered from or showing prophylactic generation in the mammal of any symptom of this disease, for example prevents the appearance of clinical symptoms; (2) suppress disease, for example make disease stop development or (3) alleviation disease, disease symptoms is disappeared.Suppressing tumor growth comprises and slows down or stop growing and make tumor regression.The effective dose of treatment disease refers to when giving the mammal that need treat and can be enough to reach amount to this treatment of diseases effect by above-mentioned requirements.IGF-IR antagonist of the present invention and PDGFR alpha-2 antagonists can use separately, also can unite to use or unite use with other one or more antitumor drug (for example chemotherapy or radiotherapy medicine).

[0044] in one embodiment of the invention, may need to measure the expression of IGF-IR in the tumor of being treated and/or PDGFR α.In this case, can collect tumor living tissue and use the conventional method in present technique field to analyze.In another embodiment of the present invention, generally express in the tumor of corresponding receptor at certain type be activated or disease carry out being bound in the process being expressed or the basis that is activated on give IGF-IR antagonist or PDGFR alpha-2 antagonists.

[0045] the IGF-IR antagonist can be that antagonist and the antagonist that may use are a kind of incessantly in outer antagonist of born of the same parents or the born of the same parents.The outer antagonist of born of the same parents includes, but are not limited to the protein or the other biological molecule that can combine with IGF-IR or one or more IGF-IR parts (for example IGF-I and IGF-II are the native ligands of IGF-IR).In one embodiment of the invention, the outer antagonist of born of the same parents suppresses combining of IGF-IR and its part.In one embodiment, described antagonist is a kind of anti-IGF-IR antibodies, for example IMC-A12.In another embodiment, described antagonist be can with the bonded soluble ligand of IGF-IR fragment.The IGF-IR antagonist can be a biomolecule in the born of the same parents, but usually is micromolecule.Such example includes but not limited to tyrosine kinase inhibitor AG1024 (Calbiochem), insulin-like growth factor-I receptor inhibitors of kinases NVP-AEW541 (Novartis) and insulin like growth factor-1/Insulin receptor INSR inhibitor B MS-554417 (Bristol Myers Squibb).Should be understood that available micromolecule is the inhibitor of IGF-IR among the present invention, but do not need that IGF-IR is had complete specificity.

[0046] anti-IGF-IR antibodies used according to the invention has one or more of following character.

[0047] 1) described antibody combines with the extracellular domain of IGF-IR and suppresses combining of IGF-I or IGF-II and IGF-IR.Can use purification or membrane-bound receptor measure inhibitory action by direct binding analysis method.In this embodiment, the preferential bonded intensity of antibody described in the present invention or their fragment and IGF-IR at least should be suitable with the native ligand (IGF-I and IGF-II) of IGF-IR.

[0048] 2) described antibody can in and IGF-IR.Part (for example IGF-I and IGF-II) and combining of IGF-IR extracellular domain can be activated the autophosphorylation that its β subunit and downstream signal transduction molecule comprise MAPK, Akt and IRS-1.

[0049] neutralization of IGF-IR is comprised inhibition, reduction, deactivation and/or destroy one or more these activity relevant with signal transduction.Can use for example interior, the earlier external back of cellular component detection bodies body interior (ex vivo) or the external neutralization of tissue, cultured cell or purification.Neutralization comprises the inhibition to IGF-IR/IR heterodimer and IGF-IR homodimer.Therefore, the neutralization of IGF-IR is had multiple effect, comprise inhibition, reduction, deactivation and/or destroy growth (propagation and differentiation), angiogenesis (mobility of (revascularization, invasion and attack and transfer) and cell and transfer (cell adhesion and aggressivity)

[0050] be inhibition to a standard of measurement of IGF-IR neutralization to the tyrosine kinase activity of this receptor.Can use common method to measure the inhibition of tyrosine kinase, for example by measuring recombinate the autophosphorylation level of kinases receptors and/or the phosphorylation of natural or synthetic substrate.Therefore, analysis of Phosphorylation is very useful for the neutralization of measuring antibody under background of the present invention.For example can use and a kind ofly phosphotyrosine is had specific antibody in ELASA detection method or western blotting, measure phosphorylation.Panek etc., J.Pharmacol.Exp.Thera.283:1433-44 (1997) and Batley etc., Life Sd.62:143-50 (1998) has described the assay method of some tyrosine-kinase enzyme activities.Antibody of the present invention can with the cell of part reaction in the tyrosine phosphorylation of IGF-IR reduce at least 75%, preferred 85%, more preferably be at least about 90%.

[0051] neutral another standard of measurement of IGF-IR inhibition that is IGF-IR downstream substrate phosphorylation.Correspondingly can measure the phosphorylation level of MAPK, Akt or IRS-I.It is about 40% that phosphorylation reduces at least, can reach at least about 60% or at least 80%.

[0052] method that in addition can the applying detection protein expression is measured the IGF-IR neutralization, and wherein detected albumen is regulated by the IGF-IR tyrosine kinase activity.These methods comprise that immunohistochemistry (IHC) method detects protein expression, fluorescence in situ hybridization method (FISH) detects gene amplification, competitive radioligand binding assay, the solid-phase matrix engram technology is western blotting and southern blotting technique method for example, reverse transcriptase polymerase chain reaction (RT-PCR) and ELISA.See below with reference to document: Grandis etc., Cancer, 78:1284-92 (1996); Shimizu etc., Japan J.Cancer Res., 85:567-71 (1994); Sauter etc., Am.J.Path., 148:1047-53 (1996); Collins, GHa 15:289-96 (1995); Radinsky etc., Clin.Cancer Res.1:19-31 (1995); Petrides etc., Cancer Res.50:3934-39 (1990); Hoffmann etc., Anticancer Res.17:4419-26 (1997); Wikstrand etc., Cancer Res.55:3140-48 (1995).

[0053] also can use earlier external back body inner analysis to measure the IGF-IR neutralization.For example can use the inhibition of being observed receptor tyrosine kinase by the activated cell line of the part of receptor by short splitting analysis method under the situation of inhibitor having inhibitor and do not exist.(US mode culture collection center (ATCC), Rockville are exactly to express IGF-IR and can be by IGF-I or the activated cell line of IGF-II as mentioned above MD) to the MCF7 breast cancer cell line.Another method relates to the growth inhibited of expressing the cell of IGF-IR after the tumor cell of detect expressing IGF-IR or the transfection.Also can use tumor model to observe inhibitory action, for example human tumor cells is injected into mice.

[0054] antibody that relates among the present invention is not limited to the neutral any concrete mechanism of IGF-IR.Anti-IGF-IR antibodies of the present invention is can born of the same parents outer in conjunction with the IGF-IR cell surface receptor, and block ligand is in conjunction with (for example IGF-I and IGF-II) with afterwards by receptor binding tyrosine kinase mediated signal transduction and prevention IGF-IR and the proteinic phosphorylation of signal transduction cascade middle and lower reaches.

[0055] 3) described antibody can be reduced IGF-IR.The quantity of cell surface IGF-IR depends on generation, internalization and the degraded of receptor protein.The quantity of cell surface IGF-IR can by detect this receptor or with the internalization indirect determination of the bonded molecule of this receptor.For example, can be by making the cell of expressing IGF-IR and being labeled antibody and contacting and measure the receptor internalization.Membrane-bound receptor is broken away from, collect and counting.By with lysis and detect and be labeled TPPA internalization antibody in the lysate.

[0056] another kind of method be use anti-IGF-IR antibodies or other materials treat after the direct quantity of mensuration cell surface receptor.For example can be colored the IGF-IR expression of cell surface by fluorescence excitation cell sorting assay.With staining cell at 37 ℃ of following incubations.Detect the fluorescence intensity of different time.A part that is colored cell can be placed 4 ℃ of incubations (no receptor internalization under this condition) in contrast.

[0057] can use a kind of different antibody to detect and measure the IGF-IR of cell surface, this antibody has specificity to IGF-IR and can not block or compete the combination of detected antibody.(Burtrum waits .Cancer Res.63:8912-21 (2003)) can reduce the IGF-IR of cell surface with the cell of Antybody therapy expression IGF-IR of the present invention.In a preferred embodiment, behind Antybody therapy of the present invention, it is about 70% that this reduction can reach at least, preferably at least about 80%, and more preferably at least 90%.Being as short as in time of four hours, can be observed remarkable reduction.

[0058] another standard of measurement of descending adjusting is the minimizing of all receptor proteins in the cell, and this standard of measurement has reacted the degraded of inner receptor.Therefore, can cause the minimizing of the total IGF-IR amount of cell with antibody treatment cell of the present invention (especially cancerous cell).In a preferred embodiment, this reduction can reach at least about 70%, preferably at least about 80%, more preferably at least about 90%.

[0059] to human experimenter's treatment, according to the preferred humanized's antibody of the present invention.Described antibody also can come from inhuman primates or humanized or chimeric antibody.In one embodiment of the invention, anti-IGF-IR antibodies can have one, two, three, four, five and/or six complementary determining regions (CDRs), and these complementary determining regions are selected from: SEQ IDNO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:45, SEQ ID NO:47 and SEQ ID NO:49 (these sequences lay respectively at CDR1H, CDR2H, CDR3H, CDR1L, CDR2L, CDR3L).In another embodiment, anti-IGF-IR antibodies can comprise one, two, three, four, five and/or six complementary determining regions (CDRs), and these complementary determining regions are selected from: SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:55, SEQ ID NO:57 and SEQ ID NO:59 (these sequences lay respectively at CDR1H, CDR2H, CDR3H, CDR1L, CDR2L, CDR3L).Antibody of the present invention (or its fragment) preferably contains the heavy chain CDRs of SEQ ID NO:35, SEQ ID NO:37 and SEQID NO:39.Perhaps these antibody (comprising its fragment) preferably contain the light chain CDRs of SEQID NO:45, SEQ ID NO:47 and SEQ ID NO:49 or SEQ ID NO:55, SEQID NO:57 and SEQ ID NO:59.A kind of antibody that human IgG l antibody I MC-A12 (WO2005016970) comes to this contains heavy chain variable domain that useful SEQ ID NO:41 represents and the light chain variable territory of representing with SEQ ID NO:51.Another preferred human antibody is IMC-2F8 (WO2005016970), contains the heavy chain variable domain identical with IMC-A 12 and is the light chain variable territory of representative with SEQ ID NO:61.Available antibody also comprise the anti-IGF-IR antibodies that can combine IGF-IR with MC-A12 or IMC-2F8 competition and can with the bonded antibody of other antigenic determinants (for example can with other antigenic determinants in conjunction with and show foregoing part blocking-up, character such as receptor internalization but not with the antibody of IMC-A12 or IMC-2F8 competition).

[0060] according to the present invention, the PDGFR alpha-2 antagonists also can be used for the treatment of.The PDGFR alpha-2 antagonists can be an antagonist in outer antagonist of born of the same parents or the born of the same parents, and can use more than a kind of antagonist.The outer antagonist of born of the same parents include but not limited to the protein that combines with PDGFR α or its one or more its parts or biomolecule (for example PDGF-AA ,-AB ,-BB ,-CC).In one embodiment of the invention, the outer antagonist of born of the same parents suppresses combining of PDGFR α and its part.In one embodiment, described antagonist is anti--PDGFR Alpha antibodies, for example IMC-3G3.In another embodiment, described conjugated protein be can with the bonded soluble ligand of PDGFR α fragment.The IGF-IR antagonist can be a biomolecule in the born of the same parents, but usually is micromolecule.In one embodiment, the PDGFR alpha-2 antagonists is AG1296 in the described born of the same parents.AG1296 (Calbiochem) is a kind of inhibitor of PDGF α, PDGF β s and c-KIT, also can react with Flt3.Other micromolecule that act on PDGFRs comprise STI-571 (imatinibmesylate, Gleevec

, Novartis) and SU11248 (sunitinib malate, SUTENT

, Pfizer).

[0061] in one embodiment of the invention, anti--PDGFR Alpha antibodies can comprise one, two, three, four, five and/or six complementary determining regions (CDRs), and these complementary determining regions are selected from: SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQID NO:10, SEQ ID NO:12 and SEQ ID NO:14 (laying respectively at CDR1H/CDR2H, CDR3H, CDR1L, CDR2L, CDR3L).Antibody of the present invention (or its fragment) preferably has the CDRs of SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6.Antibody perhaps of the present invention and fragment thereof preferably have the CDRs of the SEQ ID NO:14 of SEQ ID NO:10, SEQ IDNO:12.The aminoacid sequence of CDRs sees Table 1.

[0062] in another embodiment, anti-PDGFR Alpha antibodies or its fragment have people's variable region of heavy chain of SEQ IDNO:8 and/or people's variable region of light chain of SEQ ID NO:16.Come to this a kind of antibody and illustrating in the present invention of IMC-3G3.

[0063] antibody of the present invention or its fragment preferred in and PDGFR α.Part such as PDGF-AA, PDGF-AB, PDGF-BB or PDGF-CC can activate the cytoplasmic tyrosine kinase territory of the dimeric formation of this receptor, autophosphorylation, activation this receptor inside with combining of PDGFR α extracellular domain and start and participate in regulating DNA synthetic (gene activation) and cell cycle progression or transduction of splitted multiple signal and trans-activation approach.Anti-PDGFR Alpha antibodies usually can block ligand in conjunction with and/or receptor dimerization and suppress one or more autophosphorylations, activate the conduction of lysine kinase activity and signal.Of the present invention resisting-PDGFR Alpha antibodies can combine and stop the combination of PDGFR alpha ligands specifically with the outer ligand binding region of the born of the same parents of PDGFR α.This resisting-PDGFR Alpha antibodies or its fragment are preferably identical with PDGFR α native ligand at least with the adhesion of PDGFR α.Another kind of situation or in addition, described antibody can specificitys and a certain regional combination of acceptor monomer, otherwise this zone will form dimeric interface.Though may block or combining of block ligand and acceptor monomer not, this antibody can be blocked dimeric formation.

[0064] as above described to anti-IGF-IR antibodies, can by in the various bodies, in the body of earlier external back and in vitro method measure the receptor neutralization.In one embodiment of the invention, anti--PDGFR Alpha antibodies can reduce about 75% at least with PDGFR α phosphorylation.In other embodiments, phosphorylation has reduced about 85% or at least about 90% at least.In one embodiment of the invention, because PDGFR alpha signal transduction is suppressed, phosphorylation or downstream signal transduction pathway element (for example Akt, p42/p44 etc.) are reduced about 40% or at least about 60% or at least about 80% at least.Can use specific ligand (for example PDGF-AA ,-AB ,-BB ,-CC), the mixture of these parts or comprise PDGFs and preparations such as bone marrow aspiration liquid that other activate somatomedin are measured the receptor neutralization.

[0065] neutralization of PDGFR α comprises inhibition, reduction, deactivation and/or the destruction to one or more and signal transduction related activity.Therefore, in and PDGFR α have multiple effect and comprise inhibition, reduction, deactivation and/or destruction growth (propagation and differentiation), vascularization (revascularization, invasion and attack and transfer), cell mobility and transitivity (cell adhesion and aggressive).

[0066] body interior detection in the external back of aforesaid elder generation also can be applied to measure the neutralization of PDGFR α.For example, people SKLMS-smooth muscle oncocyte (US mode culture collection center (ATCC), Rockville, MD; ATCC HTB-88 ^TM) or by activated U118 glioma cell (the ATCC HTB-15 of PDGF-AA ^TM) can be used to analyze PDGFR α and suppress.Can determine growth inhibited with the human tumor cells of the PDGF-B expression R α that is injected into the SCID mice.

[0067] the present invention is not subjected among any PDGFR α and the restriction of mechanism.Of the present invention resisting-PDGFR Alpha antibodies combines with the extracellular domain of PDGFR α cell surface receptor, block ligand is in conjunction with (for example PDGF-AA, PDGF-AB, PDGF-BB, PDGF-CC), the phosphorylation that suppresses PDGFR α suppresses by tyrosine kinase mediated signal transduction of receptor relationship type and the activity of regulating downstream signal transduction element.Described receptor-antibody complex also can be caused the cell surface receptor downward modulation by cell internalizing and degraded.The matrix metalloproteinase that plays a role in tumor cell invasion and transfer process also can be reduced by antibody of the present invention.And antibody of the present invention may demonstrate inhibitory action to the generation and the angiogenesis of somatomedin.

[0068] as mentioned above, PDGFR alpha-2 antagonists of the present invention can be used for treating osteocarcinoma, comprises the bone metastatic carcinoma.Other PDGF-B expression R α also can include, but are not limited to matter cancer, renal cell carcinoma, tumor of prostate and soft tissue sarcoma between ovarian tumor, breast carcinoma, pulmonary carcinoma, hepatocarcinoma, gastrointestinal tract according to the tumor that the present invention is treated.Soft tissue sarcoma is from undertissue: fat, muscle, nerve, tendon, blood vessel and lymphoid tissue.Generally, described tumor tissues overexpression PDGFR α can use the expression of histochemical method for example or RNA assay PDGFR α.For example can show that to U118 cell and the bonded scatchard analysis of SKLMS-I tumor cell the PDGFR α quantity of cell surface is respectively 500 and 2500 to radiolabeled IMC-3G3.

[0069] the PDGFR alpha-2 antagonists plays a role through the alpha mediated signal transduction of the PDGFR of tumor cell oneself expression or the PDGFR α that expresses by stromal cell around suppressing (otherwise these cells can by the activation of the PDGFR α paracrine of tumor cells expression) by suppressing.Therefore, for example such antibody and other PDGFR alpha-2 antagonists of IMC-3G3 can be used for treating the tumor with PDGFR α autocrine and/or paracrine activation characteristic.

[0070] antibody fragment among the present invention can be by division complete antibody or the described segmental DNA preparation of expression coding.Can use the method for describing in the following article to prepare antibody fragment Lamoyi etc., J.Immunol.Methods, 56:235-243 (1983) and Parham, J.Immunol.131:2895-2902 (1983).This fragment may comprise one or two Fab fragment or F (ab ') ₂Fragment.This fragment also may comprise single-chain fragment variable region antibody, i.e. scFv, double antibody or other antibody fragments.The method that open these function equivalents of preparation are all arranged in the following patent: PCT application number WO 93/21319, European Patent Application No. .EP239400; PCT application number WO 89/09622; European Patent Application No. EP 338745; With European Patent Application No. EP 332424.

[0071] is used for the host cell preferred mammal cell that carrier transformed and expressed antibody of the present invention, COS-7 cell for example, the cell line in Chinese hamster ovary (CHO) cell and lymph sample source, for example lymphoma, myeloma (for example NSO) or hybridoma.Also can use for example yeast of other eucaryon hosts.

[0072] in yeast, expresses gene construct if desired, a kind ofly be applicable to that zymic selection gene is the trpl gene among the plasmid YRp7, see Stinchcomb et al.Nature, 282:39 (1979); Kingsman etc., Gene, 7:141 (1979).The trpl gene can provide selected marker for the mutant yeast strain that can not grow in tryptophan, for example ATCC numbering 44076 or PEP4-1. see Jones, Genetics, 85:12 (1977).After being compromised, trpl gene in the yeast host cell genome can provide a kind of effective ways that detect conversion by the growth in the deficiency of tryptophan environment.Equally, can be with known plasmid and Leu2 defective yeast strains (ATCC 20,622 or 38, the 626) complementation that contains the Leu2 gene.

[0073] according to the known method in present technique field can contain assimilable carbon source (carbohydrate is glucose nuclear lactose for example), (aminoacid, polypeptide, protein or its catabolite be peptone for example for nitrogenous source, ammonium salt or other) and the fluid matrix of inorganic salt (analog sulfate, the carbonate of phosphate and/or sodium, potassium, magnesium and calcium) in cultivate by transformed host cells.Described substrate also can comprise for example trace element (as ferrum, zinc, magnesium and other) of growth promoting substance.

[0074] can from the phage display library that comes with the chain variable region gene structure by people's heavy chain, separate resisting-PDGFR α and anti-IGF-IR antibodies of high-affinity according to the present invention.For example, a variable region of the present invention can obtain from the peripheral blood lymphocyte of the variable region gene that contains rearrangement.Or obtain Variable Area part for example CDR and FW district and recombinate from difference source.In addition, Variable Area part (for example FW district) can also be the consensus sequence of synthetic.

[0075] antibody of the present invention and antibody fragment can obtain from the phage display library of for example naturally occurring antibody or Fab or scFv.Should be understood that for from comprising V by one _HAnd V _LObtain single domain antibody in the antibody in territory, some aminoacid outside the CDRs should be replaced to strengthen combination, expression or dissolubility.For example, be necessary to be buried in V to those _H-V _LAmino acid residue in the interface is modified.

[0076] in addition, antibody of the present invention and antibody fragment can pass through standard hybridoma technology (Harlow ﹠amp; Lane, ed., Antibodies:A Laboratory Manual, Cold SpringHarbor, 211-213 (1998) is here as reference citation) use and can produce the transgenic mice of human normal immunoglobulin's gamma heavy chain and κ light chain (for example from Medarex, San Jose, the KM mice of Calif.) obtain.In a preferred embodiment, the genomic pith of producing people's antibody is inserted in the genome of mice and make its endogenous murine antibody under production.With these mices of the subcutaneous immunity of PDGFR α (S.C) (using complete Freund's adjuvant usually), reinforced immunological under the situation of needs.Immunization method is present technique field method commonly used.

[0077] is used to identify the preferred IGF-IR of albumen of IGF-IR binding antibody among the present invention, more preferably the extracellular domain of IGF-IR.Be used to identify the preferred PDGFR α of protein of PDGFR α binding antibody among the present invention, more preferably the extracellular domain of PDGFR α.These extracellular domains can be freely also can combine with other molecules.

[0078] the present invention also provides the foregoing antibody of coding or its segmental polynucleotide.The details of IMC-A12 anti-IGF-IR antibodies are disclosed among the WO2005016970.Table 2 is the nucleotide sequence of MC-3G3.

[0079] can or all derive from the DNA (rather than CDRs gene) of corresponding people's antibody regions and the gene of the coding CDRs in people source (be used for the SEQ ID NOS:1,3 and 5 of heavy chain variable domain CDRs, and the SEQ ID NOS:9,11 and 13 that is used for the CDRs in light chain variable territory) obtain the to encode DNA of human antibody by the most of of reorganization coding human constant region and variable region.

[0080] the suitable source of encoding antibody fragment DNAs comprises the cell of any expression full length antibody, for example hybridoma and splenocyte.As mentioned above, these fragments self can or reassemble into equivalent as the antibody equivalent.The described DNA in this part disappearance and reorganization can be implemented according to known method, the method for describing in the publication of enumerating more than for example about antibody equivalent and/or other recombinant DNA standard techniques (as described below), the method that for example describes below.Known to this professional skill field personnel, the source of another DNAs is the single-chain antibody of being produced by phage display library.

[0081] in addition, the present invention also provides the expression vector that is connected in expressed sequence, promoter and enhancer sequence that contains foregoing nucleotide sequence and operability.Developed many can be in protokaryon effective synthetic antibody polypeptide expression carrier, for example antibacterial and including, but not limited to yeast and mammalian cell cultivating system in interior eucaryon system.Carrier of the present invention can comprise the fragment of chromosome, non-chromosome and synthetic DNA sequence.

[0082] can use any suitable expression vector.Procaryotic clone carrier for example comprises the plasmid of E.coli, for example colEl, pCR1, pBR322, pMB9, pUC, pKSM and RP4.Prokaryotic vector also comprises for example derivant of MI3 and other thread single stranded DNA phagies of people of phage DNA.For example in yeast, can use 2 μ plasmids.Be useful in the carrier of expressing in the mammalian cell and comprise the SV40 derivant that is widely known by the people, adenovirus comes from retroviral DNA sequence, will derive and next shuttle vector and function plasmid and phage DNA after the aforesaid function mammalian cell carrier reorganization.

[0083] carrier for expression of eukaryon (for example, PJ.Southern.P.Berg, J.MoI.Appl.Genet., I, the 327-341 (1982) that also has other present technique field personnel to know; Subramani etc., MoI.Cell.Biol., 1:854-864 (1981); Kaufmann and Sharp, ＂ Amplification And Expression of Sequences Cotransfected with aModular Dihydrofolate Reductase Complementary DNA Gene (amplification and the expression of module dihydrofolate dehydrogenase complementary DNA gene co-transfection sequence), ＂ J.MoI.Biol.159,601-621 (1982); Kaufmann and Sharp, MoI.Cell.Biol.159,601-664 (1982); Scahill et al., ＂ Expression And Characterization Of TheProduct Of A Human Immune Interferon DNA Gene In Chinese HamsterOvary Cells (expression and the feature of a kind of people's immune interferon DNA gene product in Chinese hamster ovary cell) ＂ Proc.Nat ' l Acad.Sci.USA 80,4654-4659 (1983); Urlaub and Chasin, Proc.Nat ' l Acad.Sci.USA 77,4216-4220, (1980)).

[0084] available expression vector comprises an expression control sequenc that links together with DNA sequence to be expressed or fragment operability at least among the present invention.This control sequence is inserted described carrier controls and regulates by the expression of cloned dna sequence.Available expression control sequenc comprises: the lac system, the trp system, the tac system, the trc system, the main operator and the promoter region of phage, the control zone of fd coat protein, zymic glycolysis promoter (for example 3-phosphoglycerate kinase promoter), yeast acid p'tase promoter (for example Pho5), yeast α mating factor promoter and derive from polyoma, adenovirus, the promoter of retrovirus and simian virus (for example early stage and late promoter of SV40) and other the known sequences that can control the expression of protokaryon or eukaryotic cell and its virus or their combination.

[0085] the present invention also provides the recombinant host cell that contains foregoing expression vector.Antibody of the present invention can be expressed in cell line rather than in the hybridoma.The nucleotide that contains polypeptide chain sequence in the code book invention can be used for transforming suitable mammalian host cell.

[0086] pollutes standard screening preferred cell systems such as minimum according to high expressed amount, associated protein constructive expression with from host protein.Can be used as mammalian cell that the host is used to express and tie up to this professional skill field and be widely known by the people, comprise many immortal cell lines, such as, but be not limited to NSO cell, Chinese hamster ovary (CHO) cell, baby hamster kidney cell (BHK) and other.Other eukaryotic cells that are fit to comprise yeast and other funguses.The prokaryotic hosts that is suitable for comprises: escherichia coli (for example escherichia coli SG-936, escherichia coli HB 101, escherichia coli W3110, escherichia coli X1776, escherichia coli X2282, escherichia coli DHI and escherichia coli MRCl), Rhodopseudomonas Pseudomonas, Bacillus (for example bacillus subtilis) and streptomyces.

[0087] these recombinant host cells can be used for producing antibody or its fragment, by carrying out cell culture and antibody purification or its fragment from host cell or host cell culture medium on every side under the condition that allows antibody or its fragment expression.The sequence that can guide peptide by 5 ' end insertion coded signal peptide or the secretion at volume associated antibodies gene (see Shokri etc., Appl MicrobiolBiotechnol.60 (6): 654-64 (2003), Nielsen etc., Prot.Eng.10:1-6 (1997) and von Heinje etc., Nucl.Acids Res.14:4683-4690 (1986)) make antibody or its fragment at host cell interior orientation secreting, expressing.These secretion guiding peptide elements can come from protokaryon or eucaryon sequence.After having used suitable secretion guiding peptide according to said method, can make the aminoacid and the N end of a peptide species combine and guide this aminoacid to transport outward and move justacrine to culture medium to host cell cell colloid.

[0088] antibody of the present invention can merge with other amino acid residues.Such amino acid residue can be a kind of polypeptide label that has the separation and purification of helping.Also can consider to use other can make the amino acid residue of antibody target in certain organs or tissue.

[0089] in another embodiment, prepare this antibody, can express and reclaim antibody like this by the nucleotide of in transgenic animal, expressing encoding said antibody.For example can tissue specific expression antibody, help the recovery and the purification of antibody.In such embodiment, in mammary gland, express a kind of antibody of the present invention, can when lactogenic, secrete.Transgenic animal are including, but not limited to mice, goat and rabbit.

[0090] according to the operable antibody of the present invention comprise complete immunoglobulin, with the immunoglobulin fragment of antibodies and the antigen-binding proteins that contains the immunoglobulin antigen binding domain.Comprise for example Fab, Fab ' and F (ab ') with the bonded immunoglobulin of antigen ₂Also have other antibody patterns of having developed, not only kept bonded specificity and also had other useful characteristics, for example bispecific, polyvalency (more than 2 binding sites) and structure (for example having only) closely in conjunction with the territory.

[0091] single-chain antibody lacks the constant region that partially or completely lacks its corresponding complete antibody.Therefore they can overcome some and use the relevant problem of complete antibody.For example, some disadvantageous reaction can not appear taking place between CH and the other biological molecule in single-chain antibody.In addition, single-chain antibody has stronger permeability much smaller than complete antibody and than complete antibody, so just makes single-chain antibody can more effectively locate and combine with the target antigen binding site.And the relative smaller volume of single-chain antibody makes its probability that causes undesirable immune response in receiver's body less than complete receptor.

[0092] (each bar single-chain antibody contains by a V that connection peptides is covalently bound many single-chain antibodies _HWith a V _LThe territory) can connect by at least a or multiple connection peptides covalency and form the multivalence single-chain antibody, this antibody can be monospecific also can be polyspecific.Each bar chain of multivalence single-chain antibody all comprises a variable region of light chain fragment and a variable region of heavy chain fragment, and is connected by the one or more single-chain antibody of connection peptides and other.Connection peptides is made up of 15 amino acid residues at least.Maximum total number of atnino acid is about 100.

Article [0093] two, single-chain antibody can be combined to form double antibody, is also become the bivalence dimer.Double antibody has two chains and two binding sites, can be that monospecific also can be a bispecific.Each bar chain of double antibody all contains one and V _LThe V that the territory connects _HThe territory.Connection peptides between the territory is all enough short matches with the territory that prevents same chain, thereby promotes the complementary territory pairing of different antibodies chain and form two antigen binding sites again.

Article [0094] three, single-chain antibody can be combined to form three antibody, is also referred to as the trivalent trimer.Three antibody are by a V _LOr V _HThe amino-terminal end in territory and a V _LOr V _HThe one of carbon tip in territory directly constructs, for example, and without any the connecting peptide sequence.Three antibody have three Fv heads, and polypeptide chain is arranged with ring-type form from beginning to end.A kind of possible conformation of three antibody is planar, and three binding sites become 120 degree angles at grade mutually.Three antibody can be monospecific, bispecific or tri-specific.

[0095] therefore, antibody of the present invention and fragment thereof include, but are not limited to natural antibody, bivalence fragment for example (Fab ') ₂, unit price fragment for example Fab, single-chain antibody, strand Fv (scFv), single domain antibody, multivalence single-chain antibody, double antibody, three antibody and other and the bonded similar substance of antigenic specificity.

[0096] can by chemical reaction or biosynthetic method with anti-IGF-IR and anti--PDGFR Alpha antibodies or antibody fragment (these antibody and fragment with the cell of expressing IGF-IR (WO2005016970) or PDGFR α in conjunction with the time may be by internalization) be connected with antitumor drug.With antitumor drug that a kind of like this antibody connects comprise can destroy or damage this antibody any medicine of the tumor in bonded tumor or cell that this antibody the combines environment of living in.For example, this antitumor drug can be for example chemotherapeutics or a radiosiotope of a kind of cytotoxic drug.This professional skill field personnel understand these chemotherapeutics that is fit to, and comprise anthracycline antibiotics (for example daunorubicin and amycin), methotrexate, vindesine, neocarzinostain NCS, cisplatin, chlorambucil, cytosine arabinoside, 5-floxuridine, melphalan, ricin and calicheamycin.Use traditional method with described chemotherapeutics and described antibodies (for example seeing Hermentin and Seiler, Behring Inst.Mitt.82:197-215 (1988)).

[0097] those skilled in the art is also known the radiosiotope that can be used as antitumor drug.For example can use ¹³¹I or ²¹¹At.Use conventional art these isotopes are connected with described antibody (for example see Pedley etc., Br.J.Cancer 68,69-73 (1993)).

[0098] another selection is can activate the enzyme of prodrug to be connected with described antibody as antitumor drug.In this method, the prerequisite medicine that gives keeps inactive form until arriving target site and being converted to the cytotoxin form there always.In concrete operations, give patient's antibody-multienzyme complex and make it need be positioned the tissue regions of treatment.Give patient's prodrug then, the conversion to cytotoxic drug will take place at the tissue regions of needs treatment like this.

[0099] other antitumor drug comprises cytokine, for example interleukin II (IL-2), interleukin-4 (IL-4) or tumor necrosis factor (TNF-α).Thereby described antibody is oriented to tumor with cytokine makes cytokine performance not influence other tissue to the infringement of tumor or destruction.Use traditional recombinant DNA technology can make cytokine in dna level and described antibodies.

[100] in some embodiments of the present invention, anti-IGF-IR or anti--PDGFR Alpha antibodies and one or more antitumor drug are united use.For example conjoint therapy is seen following patent: U.S. Patent number 6,217,866 (Schlessinger etc.) (anti-EGFR-antibodies and other antitumor drug are united use); WO 99/60023 (Waksal etc.) (use is united in anti-EGFR-antibodies and radiation).Can use any suitable antitumor drug, for example chemotherapeutics, radiotherapy medicine or their combination.Described antitumor drug can be a kind of alkylating agent or a kind of antimetabolite.The example of alkylating agent includes, but are not limited to cisplatin, cyclophosphamide, melphalan and dacarbazine.The example of antimetabolite includes, but are not limited to amycin, daunorubicin and paclitaxel, gemcitabine (2,2-difluoro deoxycytidine).

[0101] available antitumor drug also comprises short division inhibitor for example taxanes, docetaxel and paclitexal.Topoisomerase enzyme inhibitor be another kind of can with the antitumor drug of antibodies among the present invention.They comprise pounces on isomerase I inhibitor and pounces on isomerase II inhibitor.Pounce on isomerase I inhibitor and comprise Irinotecan (CPT-11), amino camptothecin, camptothecine, DX-8951f and hycamtin.Pounce on isomerase II inhibitor and comprise etoposide (VP-16) and teniposide (VM-26).Estimating at present some other material pounce on the isomery enzyme inhibition activity and as the effectiveness of antitumor drug.In a preferred embodiment, the employed isomerase inhibitors of pouncing on is Irinotecan (CPT-11).

[0102] in a special embodiment, anti-IGF-IR antibodies and docetaxel are united use.In another embodiment of the invention, anti-IGF-IR antibodies and amycin are united use.

[0103] if antitumor drug is lonizing radiation, the source of lonizing radiation can be by treatment patient's external (external beam X-ray therapy-EBRT) or (brachytherapy-BT) in the body.The dosage that gives of antitumor drug depends on that multiple factor for example comprises drug type, treated the administering mode of tumor type and the order of severity and this medicine.What should emphasize is that the present invention is not limited to any concrete dosage.

[0104] described antibody (anti-IGF-IR or anti--PDGFR α) and antibody add the patient that the antitumor drug therapy also can be used for accepting complementary hormonotherapy (for example breast carcinoma) or castrates therapy (for example carcinoma of prostate).

[0105] anti-IGF-IR of the present invention and anti--PDGFR alpha-2 antagonists can be united use or be united use with other receptor antagonists that participate in the receptor of tumor growths or blood vessel generation that can neutralize.For example in one embodiment of the invention, a kind of anti-IGF-IR antibodies and a kind of resisting-PDGFR Alpha antibodies are united use.In one embodiment, target tumor cell is expressed IGF-IR and PDGFR α, and normal signal transduction element can be activated by the signal transduction through each receptor.Though the inhibition to a kind of receptor can cause generally that common downstream components activates minimizing, suppress in the time of to two kinds of receptors further to reduce activation.Some cell of tumor or surrounding tissue is significantly expressed a kind of receptor in another embodiment, and other cell is significantly expressed another kind of receptor.The growth of antagonist combination use can minimizing tumor cell and the paracrine of peripheral cell are stimulated.

[0106] bi-specific antibody can be used as another selection of drug combination.Many bi-specific antibodys that are designed to comprise various desirable characteristics are arranged.For example bi-specific antibody has minimum volume.Bi-specific antibody (each binding specificity has 2 sites) with 4 antigen binding sites has similar binding affinity with corresponding natural antibody.Some bi-specific antibody comprises the Fc district, has therefore kept the effector functions (for example, CDC (CDC) and antibody dependent cellular cytotoxicity (ADCC)) of natural antibody.Describe IgG sample tetravalent antibody among the WO 01/90192, described a kind of tetravalent antibody that comprises two double antibodies and kept effector functions among the WO2006/020258.

[0107] in another embodiment, with a kind of anti-IGF-IR antibodies or a kind of anti--PDGFR Alpha antibodies or other antagonisies and a kind of can with the bonded receptor antagonist of epidermal growth factor receptor specific (for example EGFR, Her2/erbB2, erbB3, erbB4)) unite use.Especially preferably can with the extracellular domain of EGFR combine and block one or more parts in conjunction with or in and the activated antigen-binding proteins of the inductive EGFR of part.The EGFR antagonist also comprises for example EGF, TGF-α, amphiregulin, heparin combination-EGF (HB-EGF) and beta cell regulin.Although TGF-α shows stronger short vascularization effect, EGF and TGF-α are considered to cause that EGFR-mediates activated main exogenous part.The EGFR antagonist comprises that also can suppress EGFR and other EGFR receptor subunits (being the EGFR homodimer) takes place Dimerized or with other growth factor receptorses (for example HER2) material of heterodimerization takes place.The EGFR antagonist also comprises biomolecule and micromolecule, for example can directly act on the EGFR cytoplasm domain and suppress the synthetic inhibitors of kinases of EGFR Mediated Signal Transduction.Erbitux

(cetuximab) be exactly to combine with EGFR and the bonded EGFR antagonist of block ligand.IRESSA ^TM(ZD1939) be micromolecule EGFR antagonist, it is that quinazoline derivant can be used as kind ATP inhibition EGFR.See United States Patent (USP) 5,616,582 (Zeneca Limited); WO 96/33980 (Zeneca Limited) page 4; Also as seen, Rowinsky etc., the summary of delivering in the 37th the American Society of Clinical Oncology's annual meeting in San Francisco 5, San Francisco, CA, 12-15 day May calendar year 2001; Anido etc., the summary of delivering in the 37th the American Society of Clinical Oncology's annual meeting in San Francisco 1217, San Francisco, CA, 12-15 day May calendar year 2001.Tarceva

(OSI-774) be another kind of micromolecule EGFR antagonist, it is 4-(substituted aniline) quinazoline derivant [6, two (2-methoxyl group-ethyoxyl)-quinazolines of 7--4-yl]-(3-acetylene-benzene) amine hydroxide] the EGFR inhibitor.See WO 96/30347 (PfizerInc.), it is capable for example to walk to page 4 the 34th 14-17 capable and the 19th from page 2 the 12nd.Also visible Moyer etc., Cancer Res., 57:4838-48 (1997); Pollack etc., J.Pharmacol, 291:739-48 (1999)..Tarceva

May cause the cell cycle of p27 mediation to stop by PI3/Akt and MAP (mitogen activated protein) the signal transduction of kinases approach performance function that suppresses EGFR phosphorylation and downstream thereof.See Hidalgo etc., the summary of delivering in the 37th the American Society of Clinical Oncology's annual meeting in San Francisco 281, San Francisco, CA, 12-15 day May calendar year 2001.

[0108] many micromolecule that can suppress EGFR that other have been reported are considered to that all the tyrosine kinase domain of EGFR is had specificity.About the example of micromolecule EGFR antagonist visible WO 91/116051, WO 96/30347, WO 96/33980, WO 97/27199 (ZenecaLimited) are described.WO 97/30034 (Zeneca Limited), WO 97/42187 (ZenecaLimited), WO 97/49688 (Pfizer Inc.), WO 98/33798 (Warner LambertCompany), WO 00/18761 (American Cyanamid Company) and WO00/31048 (Warner Lambert Company).The example of specificity micromolecule EGFR antagonist comprises Cl-1033 (Pfizer), it is the inhibitor of the quinazoline (N-[4-(3-chloro-4-fluoro-phenyl amino)-7-(3-morpholine-4-base-propoxyl group)-quinazoline-6-yl]-acrylamide) of tyrosine kinase, following patent is described EGFR, the 8th page of 22-26 line description of WO 00/31048 a kind of EGFR pyrrolopyrimidine inhibitor PKIl 66 (Novartis), WO97/27199 the 10th to 12 also is described this inhibitor; GW2016 (GlaxoSmithKline) is the inhibitor of a kind of EFGR and HER22; EKB569 (Wyeth) can express the growth of EFGR or HER22 cell in vivo with vitro inhibition; AG-1478 (Tryphostin) is a kind of quinazoline micromolecule that can suppress from the signal transduction of EGFR and erbB-2; AG-1478 (Sugen) is a kind of Double bottom thing inhibitor that also can Profilin matter kinase c K2; Apoptosis that the PD153035 (Parke-Davis) that can suppress EGFR enzymatic activity and tumor growth can the inducing culture cell and strengthen the cytotoxicity of cytotoxicity chemotherapeutics is in the news.SPM-924 (Schwarz Pharma) is the targeted therapy that a kind of tyrosine kinase inhibitor can be used for carcinoma of prostate; CP-546, it is the treatment that a kind of angiogenesis inhibitor can be used for solid tumor that 989 (OSI Pharmaceuticals) are in the news.ADL-681 is the targeted therapy that a kind of EGFR inhibitors of kinases can be used for cancer; PD 158780 is a kind of pyrrolopyrimidines and is in the news and can suppresses the growth of mice A4431 transplantation tumor; CP-358, the 774th, a kind of being in the news can be suppressed the quinazoline of mice HN5 graft autophosphorylation; ZD 1839 be a kind of be in the news little genus transplantation tumor model is comprised that carcinoma vulvae, NSCLC, carcinoma of prostate, ovarian cancer and colorectal carcinoma have the quinazoline of active anticancer; CGP 59326A is a kind of inhibited pyrrolopyrimidine of growth to the positive transplantation tumor of mice EGFR that is in the news; PD165557 (Pfizer); CGP54211 and CGP53353 (Novartis) are hexichol amido phthalimide (dianilnophthalimides).Natural EGFR tyrosine kinase inhibitors comprises 5,7,4 '-trihydroxy-isoflavone, herbimycin, quercetin and tri hydroxy isoflavone.

[0109] micromolecule that other have been reported can suppress EGFR and therefore also belong to the scope of the invention is the chemical compound of describing in tricyclic compounds such as the U.S. Patent number 5,679,683; Quinazoline derivant, for example U.S. Patent number 5,616, derivant described in 582 and benzazolyl compounds, for example U.S. Patent number 5,196, the chemical compound of describing in 446.

[0110] another kind can be vascular endothelial growth factor receptor (VEGFR) as the receptor that acts on target with IGF-IR or PDGFR α.In one embodiment of the invention, with a kind of anti-IGF-IR antibodies or anti--PDGFR Alpha antibodies and the use of a kind of VEGFR antagonist combination.Used in one embodiment and the bonded antagonist of VEGFR-I/FIt-I receptor-specific.In another embodiment, described VEGFR antagonist combines with the VEGFR-2/KDR receptor-specific.Especially preferably can combine with VEGFR-I or VEGFR-2 extracellular domain and (VEGF is the strongest to the activating force of VEGFR-2 by their part; PlGF is the strongest but also can be activated by VEGF to the activating force of VEGFR-I) blocking-up and/or in and part induce activated antigen-binding proteins.For example, IMC-1121 is a kind of can combine with VEGFR-2 and (WO 03/075840 with its neutral human antibody; Zhu).Another example is MAb 6.12, and it can combine with VEGFR-I solubility and that be expressed in cell surface.ScFv 6.12 comprises the V of mouse monoclonal antibody MAb 6.12 _LAnd V _HThe territory.A kind of hybridoma cell line of producing MAb 6.12 is preserved according to Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure and relevant regulations thereof, and ATCC is numbered PTA-3344.In another embodiment, described VEGFR antagonist combine with a kind of VEGFR part and block ligand to the activation of VEGFR.For example, Avastin

(bevacizumab) be exactly a kind of can be in conjunction with the antibody of VEGF.

[0111] other form relevant tumor growth factor receptor with tumor trk C (NGFR) and fibroblast growth factor acceptor (FGFR).

[0112] in the selectable embodiment of another kind, described anti-IGF-IR and anti--PDGFR Alpha antibodies can be united use with one or more ancillary drug, for example cytokine (for example IL-10 and IL-13) or other immunoactivators, for example (but being not limited to) chemotactic factor, tumor associated antigen and polypeptide.For example see Larrivee etc., ibid.Should be understood that only to give a kind of anti-IGF-IR or anti--PDGFR Alpha antibodies has been enough to prevention, suppresses or has reduced the growth of tumor process, reach effective treatment.

[0113] in a kind of conjoint therapy, before with another kind of medicine begin treatment, after the treatment neutralization treatment, give anti-IGF-IR or anti--PDGFR Alpha antibodies, also can make up at any time and give, for example before using antitumor drug therapy begin treatment and in the treatment, before, during and after before the treatment and treatment back, treatment neutralization treatment back or the treatment.For example, described antibody can begin to give in preceding 1 to 30 day in radiotherapy, and preferred the 3rd to 20 day, more preferably the 5th to 12 day.Chemotherapy can or preferably be united use with the antibody therapy while after antibody therapy in a preferred embodiment.

[0114] in the present invention, can use any suitably method or approach to give antibody of the present invention or optionally unite and give antitumor drug and/or other receptor antagonists.Comprise any the suitableeest therapy that is considered to treat patient's tumor according to the spendable antitumor drug of the present invention.Different malignant tumor requires to use specific anti-tumour antibody and specific antitumor drug, and these depend on patient's individual instances.That route of administration comprises is for example oral, intravenous injection, peritoneal injection, subcutaneous injection or intramuscular injection.The dosage of antagonist depends on many factors, comprises antagonist type for example, is treated the route of administration of the tumor type and the order of severity, antagonist.What should emphasize is that the present invention is not limited to any concrete method or route of administration.

[0115] this professional field technical staff will appreciate that the dosage of treatment and the tolerance that frequency depends on individual patient and the pharmacology and the pharmacokinetics character of employed blocker or inhibitor.We wish to reach the saturated pharmacokinetics of used medicine under the perfect condition.For example, the loading dose of anti-IGF-IR and anti--PDGFR Alpha antibodies can be for 10 to about 1000mg/m ², preferred 200-400mg/m ²Can give again several days afterwards or the additional dose in several weeks, for example 200-400mg/m ²The generation of monitor patients side effect stops treatment when serious side effects takes place.

[0116] this professional field technical staff knows also how the monitor treatment process is to determine effective dose.Shift for shifting from prostatic bone, a kind of method is the concentration of monitoring PSA.The method that other monitoring bones shift comprises bone scanning and nuclear magnetic resonance, NMR.

[0117] the above-mentioned any treatment of patient's (for example accept auxiliary hormonotherapy treatment breast carcinoma or castrate the patient of therapy for treating carcinoma of prostate) of inducing bone loss (CTIBL) can cause danger or have problems for cancer can replenish the medicine that gives to prevent CTIBL, for example diphosphate.Diphosphate comprises for example material of clodronate, Risedronate and zoledronic acid.

[0118] in whole application, various publications, articles of reference, textbook, technical manual, patent and patent are used all as a reference.These publications, patent, patent are used and the religious doctrine and the disclosure of alternative document all intactly comprise in this application more fully to describe the state of correlation technique of the present invention with reform.

[0119] is to be understood that and what predict is but that this professional field technical staff can carry out various variations these variants to the principle of the invention disclosed herein and also belongs to category of the present invention.

[0120] following examples are further illustrated the present invention, but do not limit the scope of the invention.From many publications, can obtain the detailed description of traditional means of experiment, for example the employed method of construct and plasmid, expressing antibodies and antibody fragment.These publications comprise Sambrook, J etc., (1989) molecular cloning: laboratory manual, second edition, publishing house of cold spring harbor laboratory; Coligan, J. etc. (1994) Immunology Lab guide, Wiley; Sons, Incorporated; Enna, SJ. etc. (1991) pharmacological experiment chamber guide, Wiley; Sons; Bonifacino, J.S. etc. (1999) biology laboratory guide, Wiley; Sons.Here the handbook of mentioning is all with complete comprising in this application.

Embodiment

[0121] embodiment 1

[0122] IMC-A12 and docetaxel are to the effect of tumor growth.(4) are with androgen dependent form (AI) LuCaP 35V tumor fritter (20-30mm as previously mentioned ³) distinguish subcutaneous transplantation (s.c.) to 32 6 all big castration SCID mices.Reach 150-200mm when observing transplanted tumor size ³The time, animal is divided into 4 groups at random treats research.First treated animal is accepted the treatment of 20mg/kg docetaxel.Second treated animal is accepted the treatment of 10mg/kg docetaxel.The 3rd treated animal is accepted the therapeutic alliance of 10mg/kg docetaxel and 40mg/kg A12.The 4th treated animal is accepted the therapeutic alliance of 20mg/kg docetaxel and 40mg/kg A12.All treatments are intraperitoneal administration (ip).Docetaxel administration 1 time weekly.A12 administration 3 times weekly.All animals are all accepted the treatment of 4 weeks and carried out monitoring all around before implementing euthanasia.Weekly tumor is carried out 2 times and measure, tumor size is calculated according to following formula: volume=L X W ²/ 2.According to the zoopery rules of our IACUC of University of Washington approval, when gross tumor volume reaches 1000mm ³Before or losing weight of animal surpassed original body weight 20% o'clock some animals are put to death in advance.Per two weeks weigh animal once.Carry out orbital sinus weekly and get the blood sampling.Separation of serum also uses EVIx Total PSA Assay (AbottLaboratories, Abott Park, IL) mensuration PSA level.1h injects BrdU to estimate the growth rate of interior tumor cell in tumor before mice is put to death.

[0123] will collect tumor and be divided into two parts after the mice execution.Also use the liquid paraffin embedding in the neutral buffered liquid formalin (NFB) of part tumor stuck-at-0%.The section of 5 microns sizes of preparation is also carried out immunohistochemistry (IHC) dyeing.The tumor of remainder by grind with cross 70 μ m nylon mesh separate be prepared into unicellular.

[0124] as shown in Figure 1, LuCaP 35V transplantation tumor is grown in mice rapidly, and average growth rate is 362.0 ± 72.0mm/ week under the situation of not carrying out any treatment.The animal of all non-treatment groups all must be put to death in three weeks behind the experimental group begin treatment, because this moment, their gross tumor volume all surpassed 1000mm ³When independent use 40 μ g/kg A12 treated animal, growth of tumor speed was reduced to 192.7 ± 35.6mm in the therapeutic process ³/ week.LuCaP 35V growth of tumor speed is reduced to average 29.6 ± 6.1mm when giving the docetaxel of animal 10mg/kg ³/ week.LuCaP 35V growth of tumor speed further is reduced to average 7.9 ± 1.0mm when using docetaxel and A12 therapeutic alliance ³/ week (figure Ib).The inhibition effect that docetaxel and A12 unite use can continue to treatment end the 4th week of back.If give the more docetaxel of high dose (20mg/kg) of animal, no matter whether unite use with A12, gross tumor volume does not increase in the treatment phase around scheduling to last, and on the contrary, also observing gross tumor volume has the trend that reduces.But in four week after treatment finishes, gross tumor volume further reduces in the animal groups of use docetaxel and A12 therapeutic alliance.By contrast, using the balanced growth speed of the animal groups gross tumor volume of docetaxel treatment separately is 27.0 ± 16.1mm ³/ week.These results show under given docetaxel dosage, unite and use A12 can strengthen docetaxel inhibitory action to tumor growth in therapeutic process or after the treatment.

[0125] PSA is the clinical parameter of the evaluation tumor of prostate growth generally used.Measure the Serum PSA level of animal in treatment neutralization treatment back.As scheme shown in the Ic, the animal of using A12 and docetaxel or using the treatment of 20mg/kg docetaxel separately, significant change does not take place in Serum PSA level, is suppressed consistent with tumor growth.After treatment finishes, use the animal serum PSA level of docetaxel treatment to increase separately, by contrast, the animal PSA level of use docetaxel and A12 therapeutic alliance remains unchanged even reduces.These data show that all use docetaxel and A12 therapeutic alliance can still keep the inhibitory action to tumor growth after treatment finishes.

[0126] docetaxel and anti-IGF-IR antibodies are united the use cell death inducing.Use the breach labelling technique (TUNEL) of foregoing end deoxynucleotide based transferase mediation and use Apop-Direct kit (BD BioScience) to carry out iodate third ingot (PI) dyeing and measure in docetaxel and A12 the body and act on the cell cycle of testing end point and cell survival rate.Basic process is as follows: with 1 x 10 in the single cell suspension ⁶Cell fixation and then is fixed 30 minutes with under 70% the ethanol-20 ℃ in 10% neutral buffered liquid formalin.After the flushing for several times, the Triton X-100 permeation cell with 0.1% and with the dUTP that combines FITC and end deoxynucleotide based transferase (TdT) in 37 ℃ of incubation 1h.Use PI/RNase buffer (PI of 100 μ g/ml, 50 μ g/ml RNase) incubation 60 minutes under room temperature afterwards.Use flow cytometer BD FACscan analytic sample.Use CellQuestPRO software analysis data.

[0127] treatment finishes back four week, (using the animal groups of 10mg/kg docetaxel treatment is 66.7% using docetaxel, using the animal groups of 20mg/kg docetaxel treatment is 77.8%) and the animal tumor of A12 therapeutic alliance in observe the apoptosis (figure .2b and table 1) of remarkable ratio, have nothing to do with the using dosage of docetaxel.The average originating rate of apoptosis incident is 15.0 ± 4.3% in these tumors.In the animal groups of using the docetaxel treatment separately, do not detect apoptosis.On the contrary, great majority (using the treatment group of 10mg/kg docetaxel is 88%, and using the treatment group of 20mg/kg docetaxel is 100%) tumor enters normal cell cycle (figure .2a and table 3)

[0128] control the different multiplication capacities that finish the back tumor cell for the treatment of in order further to estimate, with paraffin section with anti-BrDu antibody staining (paraffin-section of stained with anti-BrDuantibody).Tumor sample is fixed and is embedded in the liquid paraffin with 10% NBF, be cut into the thin slice of 5 μ m.After removing paraffin and rehydration, with citric acid (pH6.0) recovery antibody 2 X 5min under 95 ℃ of 0.01M.With thin slice cooling 30 minutes, then with the continuous rinse of PBS.H with 0.3% ₂O ₂The methanol solution incubation makes the endogenous peroxydase inactivation.After in the PBS solution that contains 0.05% Tween 20, sealing 1 hour with 1.5% conventional lowlenthal serum, with thin slice and mouse anti-BrdU antibody (1 μ g/ml) incubation 1 hour.Then successively with biotinylated goat-anti-mice IgG incubation 30 minutes, with avidin (the Santa Cruz Biotechnology) incubation of peroxidase labelling 30 minutes, with diaminobenzidine (DAB)/hydrogen peroxide chromogen substrate (Vector Laboratories, Burlingame, CA) incubation 5-10 minute.All incubation processes are all at room temperature carried out.Dye thin slice is counter with hematoxylin (Sigma), and seal up for safekeeping with mountant (Fisher Scientific, Fair Lawn, New Je rsey).For negative control, replace one-level to resist-BrdU antibody with mice IgG (Vector Laboratories).Observe thin slice and obtain digital image at the Zeiss microscopically.Calculate 10 of each section BrdU labeled cell check figure and total cell check figures in the visual field at random.Just can calculate the rate of increase with BrdU positive cell check figure divided by total cell check figure.Each 10 zone of thin slice counting.(Richard Allen, Kalamazoo MI) carry out H﹠amp to use h and E; E dyeing.

[0129] use is taken in BrDu in the animal of docetaxel and A12 therapeutic alliance and obviously is less than the animal for the treatment of with docetaxel separately (Fig. 3).It is consistent with above cell cycle and apoptotic observed result that these BrDu mix data, and this shows that A12 has significantly strengthened the cytotoxicity of docetaxel.

[0130] with of the comparison of the tumor of docetaxel and anti-IGF-IR-antibody combined treatment with the oncogene differential expression regulation and control of using the docetaxel treatment separately.In order to determine the potential mechanism of A12, use immunohistochemistry and flow cytometry analysis method to detect the expression of IGF-IR in all results tumors to the remarkable potentiation of docetaxel.The expression of comparing (data not shown) surperficial IGF-IR between all treatment groups or with matched group does not have difference.Use cDNA microarray analysis method detects the gene expression after the treatment in the animal tumor of accepting the treatment of 20mg/kg docetaxel and accepting 20mg/kg docetaxel and A12 therapeutic alliance.Analyze based on SAM, compare with the tumor of independent use docetaxel treatment, it is differentially expressed to identify 49 kinds of genes in the tumor of using docetaxel and A12 therapeutic alliance, this species diversity greater than 2 times and wrong discovery rate less than (FDR) 10% (data not shown).Identify 13 kinds of genes and may participate in the regulation and control (table 4) of apoptosis or cell cycle.All 13 genes exist 2 times of differences and FDR less than 0.02% at least between two kinds of treatments.Compare with the tumor of independent use docetaxel treatment, use the tumor kind of docetaxel and A12 therapeutic alliance that 9 kinds of gene downward modulations are arranged, 4 kinds of genes raise.

[00100] for the gene that is screened, described result is verified by real-time RT-PCR.The Standard PC R fragment of purification of target cDNA.This standard fragment is carried out a series of dilution, from 10ng/ μ l to 10 ^-3Pg/ μ l also carries out real-time RT-PCR drawing standard curve.Use the Superscript first chain synthetic system (Invitrogen), with each combination of 1 μ g and the total RNA of tumor sample carry out synthesizing of cDNA first chain.In the 20 μ l reaction systems that contain 1 μ l cDNA, first chain, Auele Specific Primer group and Lightcycler FastStart DNA Master Plus SYBR Green, use Roche Lightcycler to carry out real-time PT-PCR (Roche according to operation instruction, Nutley, NJ).Use Lightcycler software v3.5 that the RT-PCR product is carried out curve analysis.With agarose gel electrophoresis checking amplification amount.Each sample divides to be analyzed in duplicate.The results are shown in Figure 4.

[0131] in down-regulated gene, TUBB show the docetaxel resistance (Tanaka etc., 2004, Int.J.Cancer 111,617-26) and the increase of BIRC 5 (survivin) expression demonstrate dependency (de Angelis etc. with aggressive carcinoma of prostate and androgen therapy resistance, 2004, Int.J.Oncol.24,1279-88; Zhang etc., 2005, Oncogene 24,2474-82).In addition, TUBB be a kind of IGF-IR regulator gene and the conversion that participates in IGF-IR mediation (Loughran et al., 2005, Oncogene 24,6185-93).In four kinds of up-regulated genes, IGFBP3 shows to the inhibitory action of IGF-part signal transduction with to the effect of prostate gland cancer cell apoptosis induced.(Grimberg etc., 2000, J.Cell.Physiol.183,1-9).

[0132] level of treatment back serum A12.Mensuration is accepted the animal serum A12 level of docetaxel and A12 therapeutic alliance.Finish 2 all backs serum A12 levels in treatment and descended 100 times, and detected level lower (Fig. 5) after around treatment finishes.

[0133] total cytotoxicity.Measure the cytotoxicity that docetaxel and IMC-A12 unite use.Though the cross reactivity of A12 and Mus IGF-IR, accept combination medicine treatment greater than 95% or accept docetaxel separately the animal of treatment compare with the contrast tumor animal and do not observe abnormal day's activities or behavior.The remarkable effect (data not shown) to kidney cell is not all observed in cell cycle and apoptotic analysis in any treatment group.Do not observe the notable difference (Fig. 6) of body weight between the treatment group.

[0134] anti-IGF-IR antibodies therapy for treating bone shifts.Prostate gland cancer cell is directly injected the tibia of SCID mice and estimate the anti-IGF-IR antibodies therapy the therapeutic effect of prostate gland cancer cell at the bone transforming growth.Can directly generate metastatic tumo(u)r and need not depend on by this method and attack from circulation chemotactic dependency.Many cell lines all can be used for setting up bone and shift.These comprise PC-3, LuCaP35 and the LnCaP cell that can cause osteolytic lesion, can cause into LuCaP 23.1 cells of bone lesion in addition.

[0135] LuCaP 23.1 cells can be expressed IGF-IR, and the survival rate in the bone environment is for～80% and cause osteoblastic reaction.In preliminary experiment, the tumor tibia with the contrast tibia compare, the LuCaP23.1 cell sample show bone volume and tissue volume ratio remarkable increase (254-503% of contrast, p=0.024).Newborn bone trabecula all appears in all LuCaP 23.1 tumors in the tibia, but does not occur in the normal specimens, also has a large amount of tumor points to replace normal bone marrow.The growth of tumor and bone extends to outside the raw bone in some samples.Also observing LuCaP 23.1 sample %BV/TV after castrating increases; The 212-354% (p=0.024) of the tibia %BV/TV of tumor growth for no tumor tibia arranged.The observed result of 23.1 grafts of LuCaP in the tibia shown exist the activated bone of tumor cell from the beginning to form.And these tumors show the similarity that many and human osteoblast cell shift sample, comprise the increase of a large amount of tumor points and mineralising bone quantity.

[0136] in order to estimate the effectiveness of IMC-A12 therapy, also per two weeks of SCID mice is gone in LuCaP 23.1 tumour transplatations detect Serum PSA level to estimate tumor growth.Fortnight is castrated all animals before transplanting the tibia tumor cell, gives experiment mice IMC-A12 when (show and formed tumor) when Serum PSA level reaches 5-10ng/ml.Time subcutaneous injection 40mg/kg IMC-A12 continued for 6 weeks on every Wendesdays.

[0137] use in the scope of the two tumor cell injection site 2.5mm x 2.5mm of X-ray absorption photometry (PIXImus Lunar photodensitometer) when transplantation tumor or the correspondence position of offside tibia is measured the bone mineral density of tumor tibia and tumor free offside tibia.Estimate once infringement by measuring per two weeks of Serum PSA level.After matched group is being castrated according to Serum PSA level (LuCaP 35〉60ng/ml, LuCaP 23.1〉500ng/ml) judge taken place once more that bone infringement, the photography of X line demonstrate that bone damages or animal when being worse off (comprimise) with all sacrifice of animal.Putting to death animal injection in previous hour BrdU with the monitoring tumor cell proliferation.Put to death the BMD that carries out radiograph (FaxitronX-ray MX-20) before and when putting to death, measure the both sides tibia.

[0138] accepting the mice serum PSA level significantly lower (Fig. 7) of IMC-A12 treatment and the BMD relevant with the skeletonization metastatic tumour increases and remarkable reduction (table 5) also occurred.The BMD measurement result of no tumor tibia shows that the IMC-A12 therapy does not cause the reduction (osteoporosis) of bone density.The X-ray photograph of accepting the IMC-A12 treatment mice and the mice of not receiving treatment shows that the tumor progression of the mice of receiving treatment is significantly reduced or suppresses (Fig. 8).

[0139] anti-IGF-IR antibodies and docetaxel therapeutic alliance bone shift.The SCID mice is castrated before two weeks at the tibia tumor injection.LuCaP 23.1 prostate gland cancer cells are injected directly into the mice tibia shift, thereby increase the skeletonization infringement to generate bone.This shifts thing and expresses IGF-IR.When reaching 5-10ng/ml, Serum PSA level shows that tumor forms, and is divided into 4 groups at random with animal then.

[0140] two treated animal is three 40mg/kg IMC-A12 of lumbar injection weekly, continued for six weeks, one group of IMC-A12+20mg/kg docetaxel of lumbar injection and continued for six weeks weekly wherein, another is organized weekly three IMC-A12+10mg/kg docetaxels of lumbar injection and continued for six weeks.Matched group lumbar injection 10 or 20mg docetaxel and do not use IMC-A12.

[0141] monitors the PSA measured value of animal weekly.After treatment finished, the PSA measured value that animal is monitored in continuation weekly tumor regrowth occurred until the animal groups of independent use docetaxel treatment.Use separately the animal groups PSA level of docetaxel treatment to rise (though slower), accept that the PSA level stops to rise in the mice of IMC-A12+ docetaxel treatment, even in some animal, begin to descend than the speed of not treating animal groups.After observed result shows that the PSA level continues reduction even continue to the treatment of six weeks to finish.

[0142] as implied above, when transplantation tumor and execution animal, measure BMD, and before putting to death, carry out the X line and take pictures.Accept the very little or not increase of animal groups BMD increase that the IMC-A12+ docetaxel is treated, the very little or nothing of the osteoblast activity that the X skiagram shows.

[0143] anti-IGF-IR antibodies and docetaxel therapeutic alliance bone shift.Mechanical digestion LuCaP23.1 human prostate tumor fritter (20-30mm ³).With 2-5 x 10 ⁵Great-hearted LuCaP 23.1 injection cells are gone in all big SCID mice tibias of 6-8.21 mices are divided into three groups at random to be studied.Serum PSA level of each week monitoring after the injection tumor cell.When reaching 5-10ng/ml, Serum PSA level shows that tumor begins growth.First group is used the contrast normal saline buffer solution.Around second group 20mg/kg docetaxel of lumbar injection also continues weekly.Around the 3rd group three 40mg/kg docetaxels of lumbar injection also continue weekly.When all treatments finish with Dexa-scanning and X-ray measuring BMD to determine that treating the effect that causes is skeletonization or molten bone.

[0144] Serum PSA level is subjected to suppress to reflect (figure .9a) and uses docetaxel separately or unite and use docetaxel and A12 can suppress not have significant difference between LuCaP 23.1 growth of tumor and two kinds of treatments.But after treatment finishes, use the animal serum PSA level of docetaxel treatment to begin to rise separately, show that tumor regrows; Yet in the animal of using conjoint therapy, observe lasting inhibition, show the prolongation of resting stage of treatment back tumor Serum PSA level.Serum PSA level shows the dependency (Fig. 9 b) with the shown bone tumor volume of bone density (BMD) and X photo.In the 5th week, the average bone density of matched group, docetaxel 20 and docetaxel 20+A12 treatment treated animal is respectively 0.112 ± 0.01,0.09 ± 0.02 and 0.05 ± 0.009 (mean value SEM).Bone density shows tangible reduction trend in treatment.

[0145] embodiment 2

[0146] bone marrow aspiration liquid is induced the Akt phosphorylation.Normal male donor's bone marrow sample is by Cambrex (Poietics ^TMDonor Program) provides.In 1,500rpm is centrifugal with sample, separates solvable and cell mutually.Supernatant is used successively the membrane filtration of 0.8 μ m and 0.22 μ m.Give the cell in the 1ml culture medium (the whole dilution factor of 1:20) with the bone marrow aspiration liquid of 50 μ l.

[0147] there is the experiment of carrying out down for serum, before being exposed to bone marrow, cell cultivated 24 hours in the DMEM that contains 10% FBS and 50 μ g/ml gentamycins.Exist for serum-free the experiment (hungry cell) carried out down with PBS with cell flushing twice, growth medium replaced with the DEME of serum-free and before adding bone marrow aspiration liquid with cell incubation 4 hours.During use, before adding bone marrow aspiration liquid, in culture medium, add a kind of pdgf receptor specific inhibitor AG-1296 (Rice etc., 1999, Amer.J.Path.155,213-21).When preliminary experiment, add IMC-3G3 antibody according to following method.

[0148] in the PC3-ML cell of expressing FDGFR α, detects the activation of bone marrow aspiration liquid, but in the DU-145 cell that lacks this receptor, do not detect Akt.In an example experiment,, cell was cultivated 4 hours in the culture medium that does not contain serum in advance for the activation of serum component to Akt minimized.Add bone marrow aspiration liquid and cause intensive Akt phosphorylation in the PC3-ML cell, but in the DU-145 cell, do not cause such effect (figure .10A).In order to estimate the significance of this effect, in the presence of serum, carry out the experiment second time.Under the condition that serum exists, also observed intensive Akt phosphorylation in the PC3-ML cell (figure .10B).And the effect that causes in the DU-145 cell is very little.

[0149] the alpha mediated Akt phosphorylation of PDGFR.Osteoblast and osteoclast are all secreted PDGF-AA and PDGF-BB, and they provide these somatomedin in the solubility environment of bone marrow.For determine the PC3-ML cell to the reactivity of bone marrow aspiration liquid whether with relevant through the signal transduction of PDGFR α, under the condition that has or do not exist 20 μ M AG-1296, in the PC3-ML cell, add bone marrow aspiration liquid.The AG-1296 of this concentration can suppress the inductive Akt of PDFG-BB fully and activate (Figure 11 A).AG-1296 to the activated inhibition of the inductive Akt of bone marrow aspiration liquid greater than 40% (Figure 11 B and D).This shows that the inductive Akt activation of transduction of PDGFR alpha signal and bone marrow has remarkable relation.

[0150] estimates PDGF-AA and the BB pair of direct effect that the PDGFR alpha signal relevant with other compositions of bone marrow aspiration liquid transduceed.After measured, in three different donor's bone marrow aspiration liquid the concentration of PDGF-AA and BB in the scope of 400pg/ml to 2ng/ml.If with 20 times of bone marrow aspiration liquid dilutions, so detected cell in fact is exposed to concentration range in 20 to 100pg/ml PDGF-AA and BB.Therefore, with the PDGF-AA of 100pg/ml and-BB handles the PC3-ML cell.The Akt phosphorylation degree is less than 10% of the inductive phosphorylation degree of bone marrow aspiration liquid.(Fig. 3 C and D) therefore, PDGFR alpha signal transduction to the activation of Akt approach may relate to PDGFR alpha ligands rather than PDGF-AA and-BB and/or other mechanism rather than by part directly in conjunction with activating PDGFR α.

[0151] a kind of resisting-PDGFR Alpha antibodies is to the inhibition of Akt phosphorylation.Antibody I MC-3G3 with neutralization has specificity to people PDGFR α, detects the inhibition of this antibody to Akt phosphorylation in the PC3-ML cell.The concentration of precincubation in 30 minutes and 20 μ g/ml can in and the stimulation of 30ng/ml PDGF-BB (figure .12A).Use this antibody to treat and also the inductive Akt phosphorylation of bone marrow can be suppressed about 40% (figure .12B and C).Find also that simultaneously IMC-3G3 depends on time of precincubation to the inhibitory action of Akt phosphorylation, 120 minutes (Figure 12 D) is more remarkable than 30 minutes (figure .12B and C) effects for incubation.A kind of explanation that this phenomenon is possible is exactly that IMC-3G3 induces PDGFR α internalization, and its inhibitory action is not only relevant with the block ligand combination but also relevant with the elimination cell-membrane receptor.

[0152] embodiment 3

[0153] the anti-PDGFR Alpha antibodies of separation and purification people.By standard hybridoma technology (Harlow ﹠amp; Lane, ed., Antibodies:A Laboratory Manual, Cold Spring Harbor, 211-213 (1998), here quoted by complete) (Medarex Inc., Sunnyvale CA) produce the anti-PDGFR Alpha antibodies of people to use the transgenic mice of expressing human immunoglobulin gamma heavy chain and κ light chain.People PDGFR α extracellular domain (ECD) is available from R﹠amp; D Systems (Minneapolis, MN).With quantity is 3 x 10 ⁷The subcutaneous immunity of porcine aorta endotheliocyte (s.c) the KM mice of stably express PDGFR α (PAE Ra).Complete Freund's adjuvant subcutaneous injection 50 μ g PDGFR α ECD and lumbar injection 3 x 10 are used in the back all around ⁷PAE Ra is with reinforced immunological.Use complete Freund's adjuvant to inject 25 μ g PDGFR α ECD and continue immunologic facilitation twice, at interval three weeks.

[0154] separation has the mouse boosting cell of high serum combination and blocking-up titre and merges with the myeloma cell.Sub-clone is cultivated the antibody that has the active hybridoma culture of blocking-up and use these hybridomas of G albumen chromatograph purification.

[0155] adhesion of evaluation IgGs and PDGFR α in direct binding analysis.PDGFR α ECD in the PBS solution is fixed in (100ng/ hole) in 96 orifice plates.Washing plate with PBST (PBS+0.05% tween) also sealed 2 hours down in 25 ℃ with PBSM (PBS that contains 3% milk, 200 μ L/ holes).To wash plate in 25 ℃ of following incubations 1 hour and with PBST with the IgGs and the immobilization PDGFR α ECD of PBSM dilution.With two anti-(sheep F (ab ') ₂(BioSource International, Camarillo CA) dilutes with 1:5000 with PBSM anti-human IgG-horseradish peroxidase conjugate, adds 1 hour down at 25 ℃.With PBST wash add behind the plate TMB peroxidase substrate (KPL, Gaithersburg, MD), with 100 μ L 1mol/LH ₂SO ₄Cessation reaction.(Molecular Devices, Sunnyvale CA) read plate at the A450 place with microplate reader.

[0156] estimates PDGF blocking-up (see Duan etc., 1991, J.Biol.Chem.266:413-8 is quoted by complete) here with solid phase PDGF blocking-up analytic process.Dilute PDGFR α ECD and be coated on (the flat 1 x 12Removawell radiation of Immulon 2HB protein binding polystyrene bar in the 96 hole microtitration plates with PBS; Dynex Technologies, Chantilly, VA).Under 25 ℃, each hole bag is continued 3 hours by 60ng PDGFR α, and cumulative volume is 100 μ L.Washing plate also spends the night in 4 ℃ of sealings with 25mmol/L HEPES (pH 7.45), 0.5% gelatin, 100mmol/L NaCl and 0.1% polysorbas20 for twice.Titer plate is warming up to 25 ℃ and kept 20 minutes, uses binding buffer liquid (25mmol/LHEPES (pH745), 0.3% gelatin, 100mmol/L NaCl, 0.01% polysorbas20) to wash plate once then.In each hole, add the IgGs of 50 μ L and in 25 ℃ of incubations 30 minutes.With binding buffer liquid dilution iodate PDGF and in each hole, add the solution of 50 μ L1nmol/L.Titer plate in 25 ℃ of incubations 2 hours, is washed 5 times with binding buffer liquid then.With gamma counter each hole is counted.According to Heldin etc., 1988, EMBO J.7, the method for describing among the 1387-93 is carried out the blocking-up analysis based on cell.

[0157] (Piscataway NJ) measures antibody and the bonded kinetics of PDGFR α for BIAcore, Inc. with BIAcore 3000 instrument.Be fixed in PDGFR α ECD on the sensing chip and inject the antibody of variable concentrations.The induction collection of illustrative plates and the service routine BIAEvaluation 2.0 that obtain under each concentration estimate to measure speed constant.According to speed constant K _Off/ K _OnRatio calculation affinity costant K _D.

[0158] Figure 13 represents that human monoclonal antibodies IMC-3G3 combines with the dose dependent of immobilization PDGFR α ECD in the ELISA experiment.Reaching maximum PDGFR α ECD is 0.06nmol/L (table 6) in conjunction with 50% antibody concentration.BIAcore instrument surface plasma body resonant vibration detects and shows ED ₅₀Consistent with the Kd of antibody.Described monoclonal antibody also can block [ ¹²⁵I] PDGF-BB combines IC with the immobilization receptor ₅₀Be 0.43nmol/L.Binding site with PDGF-AA and PDGF-BB on the PDGFR α does not structurally coexist.The epi-position of gained data show 3G3 is spatially overlapping with the binding site of two kinds of somatomedin.

[0159] activation of the inhibition of receptor phosphorylation and downstream effect device molecule.With the effect of PAE Ra raji cell assay Raji IMC-3G3 to the inductive intracellular signal transduction of PDGF.With cell inoculation to 6 hole Falcon tissue culturing plate (250000 in cell is contained in every hole) and incubated overnight.Use serum-free medium rinse and incubation then.Make cell be in resting state after the incubated overnight, handle cell with antibody in 37 ℃ and added PDGF-AA or PDGF-BB in 30 minutes then and under 37 ℃, continued incubation 10 minutes.With cell detachment and be dissolved in (50mmol/L Tris-HCl (pH 8.0), 1% Triton X-100,150mmol/LNaCl, 1mmol/L EDTA, 0.1% SDS, 1mmol/L sodium orthovanadate and protease inhibitor (Complete Mini in the lysis buffer of 200 μ L, Roche, Mannheim, Germany)).With SDS-PAGE and western blotting (using enhanced chemiluminescence reagent and Hyperfilm (AmershamBiosciences)) analysis of cells lysate.

[0160] detects the inhibition ability of antibody to the inductive receptor tyrosine phosphorylation of part.Concentration be respectively 1 and the PDGF-AA of 3nmol/L and PDGF-BB PDGFR α tyrosine phosphorylation can be strengthened 5 times.The part of higher concentration (10nmol/L) can cause receptor phosphorylation still less, and this may be because the inductive Degradation of part.Inductive receptor of PDGF-BB-that is suppressed by antibody and background level are near (Figure 14 A, top line).Induce receptor phosphorylation to obtain similar data with PDGF-AA.

[0161] PDGFs transduction mitogenic signal and by the performance of downstream effect device albumen to expressing the anti-apoptotic effect of corresponding recipient cell.Therefore, measure the inhibition of monoclonal antibody to MAPKs p44/p42 and Akt (participating in cell growth and anti-apoptosis pathway respectively).Anti--PDGFR Alpha antibodies response PDGF-BB (figure .2A) and PDGF-AA (not shown), the phosphorylation of inhibition MAPKs and Akt.Inhibition to PDGFR α phosphorylation is not a dose dependent, and 0.25nmol/L can reach 50% suppression ratio (figure .14B).

[0162] anti-short cell division activity.Measure the ability of the anti--inductive PAE Ra of PDGFR Alpha antibodies blocking-up PDGFAA cell mitogen.(1 x 10 is contained in every hole with cell inoculation to 96 hole tissue culturing plate ⁴Individual cell) and add the culture medium incubated overnight of 100 μ L in every hole.Add 75 μ L serum-free mediums with serum-free medium rinse culture hole and in each hole and make the cell serum starvation overnight.Add IgG (25 μ L/ hole) and culture plate was placed 37 ℃ of following incubations 30 minutes.Add PDGF-AA or PDGF-BB (25 μ L/ hole) and culture plate was placed 37 ℃ of following incubation 18-20 hours.In each hole, add 0.25 μ Ci[ ³H] thymidine continued incubation 4 hours with culture plate after (25 μ L/ hole).With serum-free medium dilute antibody, PDGF and [ ³H] thymidine.Use trypsin 100/xL/ hole then with the PBS flushing cell that contains 1% bovine serum albumin) handle and make cell detachment.With MACH III cell harvestor with cell harvesting to filter and with distilled water washing three times (Tomtec, Inc., Hamden, CT).Filter is handled the back go up the radioactivity that mixes of measuring DNA at scintillation counter (Wallac Microbeta, model 1450).

[0163] in the PAE of serum starvation Ra cell, adds IMC-3G3 and can mix (Figure 15), EC by the inductive thymidine of specificity inhibition PDGF-AA ₅₀Be 8.3nmol/L.This antibody can also suppress the inductive PAE Ra of 3nmol/L PDGF-BB-cell mitogen, EC ₅₀Be 1.25nmol/L (data not shown).

[0164] growth inhibited of the human tumor cell line of PDGF-B expression R α.The human tumor cell line that detects PDGF-B expression R α with measure the people anti--the PDGFR Alpha antibodies is in the body and the pernicious effect that increases of vitro system.Through cells were tested by flow cytometry, the tumor cell line of two kinds of PDGF-B expression R α is respectively SKLMS-I (leiomyosarcoma) and U118 (glioblastoma).These cell lines also can respond the part in the mitosis detection and can form tumor in mice.SKLMS-I both can activate also and can activate by autocrine by paracrine.Quantitative sandwich enzyme immunoassay (R﹠amp; D Systems) showing can PDGF-B expression-AA when SKLMS-I grows in culture medium.

[0165] after PDGF-AA stimulates the SKLMS-I cell as can be seen from Figure 16 A, causes that IMC-3G3 suppresses the phosphorylation of Akt and MAPKs.Inhibition to the Akt phosphorylation is 100%, is 80% to the inhibition of MAPKs.This antibody also can effectively suppress the phosphorylation in the U118 cell.The mitosis of the inductive tumor cell of part also is blocked.In the U118 of serum starvation cell, add anti--the inductive thymidine of PDGF-AA mixes by specificity and suppresses (Figure 17 A), EC during the PDGFR Alpha antibodies ₅₀Be 3.4nmol/L.This antibody can also suppress the mitogenic response (Figure 17 B) of the inductive SKLMS-I cell of PDGF-AA-, EC ₅₀Be 5nmol/L, and the mitogenic response (Figure 17 C) of PDGF-BB stimulation.(66nmol/L is 40% only to observe partial inhibition to the inductive mitogenic response of PDGF-BB in the U118 cell; Figure 17 D).This is because these cellular expressions PDGFR α and PDGFR β (data not shown).

[0166] inhibition that the tumour transplatation thing is grown.In nude mouse glioblastoma (U118) and leiomyosarcoma (SKLMS-I) subcutaneous transplantation model, IMC-3G3 is carried out detecting in the body.By (Wilmington MA) injects and matrigel (Collaborative ResearchBiochemicals, Bedford, MA) blended 10 x 10 for Crl:NU/NU-nuBR, Charles RiverLaboratories to female nude mouse ⁶SKLMS-1 or U118 cell carry out Subcutaneous tumor and transplant.Allow the average external volume (π/6 x extreme length x vertical width of tumor ²) reach about 400mm ³Mice is divided into 5 groups (n=12) at random and carries out twice lumbar injection weekly.First treated animal be the solvent control group (0.9% NaCl, USP for Irrigation, B/Braun).The the 2nd to 4 treated animal accept 6,20 and 60mg/kg constant anti--treatment of PDGFR Alpha antibodies.The 5th group of mice accepted the treatment of 60mg/kg human IgG (Sigma).Accept 6,20 or 60mg/kg anti--loading dose of the mice group of PDGFR Alpha antibodies or human IgG treatment is respectively 21.4,71.4 and 214mg/kg.Calculated load dosage makes according to semiweekly dosage regimen just can reach steady plasma-drug concentration since the administration first time (eliminating the half-life is 7 days).Measure gross tumor volume per two weeks and the tumor growth of treatment group is compared by repeated measure ANOVA.

[0167] shown in Figure 18 A, handle the mice of (P=0.74) compares with saline solution, human IgG is to not effect of the growth of glioblastoma, significantly suppressed growth of tumor yet dosage is the resisting of 6 (P=0.06), 20 (P=0.03) and 60 (P=0.0004) mg/kg-PDGFR Alpha antibodies.When U118 research finishes, 6,20 and the %T/C value of 60mg/kg 3G3 processed group [(mean tumour volume the during mean tumour volume of 3G3 processed group when research finishes/treatment beginning)/(mean tumour volume when mean tumour volume/treatment of control treatment group begins during the research end) x 100] be respectively 67%, 63% and 35%.And 6,20 and the 60mg/kg3G3 processed group in respectively 5 and 12 of merely hitting of 4,11 in 12 mices merely hit 11 merely hit and observe tumour regression.In all matched groups, all do not occur degenerating.

[0168] Figure 18 B shows that 6 (P=0.02), 20 (P=0.003) and 60 (P＜0.0001) treatment of mg/kg have significantly suppressed the growth of leiomyosarcoma.6,20 and the final %T/C value of 60mg/kg treatment group be respectively 66%, 57% and 31% and do not have a tumour regression.

Histological examination to graft when [0169] treatment finishes shows that there are significant difference in the animal groups tumor of accepting the contrast treatment and the animal tumor of receiving treatment.The tumor of excision is fixed 24 hours in 4 ℃ in the QDL fixative.With the thin slice that is cut into 4 μ m after the paraffin embedding, with Mayer ' s H﹠amp; E (Richard Allen, Kalamazoo, MI) sample of dyeing formalin fixed.

[0170] compares with saline solution-matched group, still less and have a loose zone of more cell (figure .18C) with great-hearted tumor cell in the U118 group of maximum dose level (60mg/kg) treatment.Compare with saline solution-matched group, the SKLMS-I graft of being treated also had the minimizing that vigor tumor cell quantity and cell are filled at the 25th day.

[0171] be activated vitro inhibition to the alpha mediated glioblastoma of PDGFR.Using anti-PDGFR Alpha antibodies or human IgG antibody to treat the phosphorylation level of estimating receptor in the U118 cell the last week.U118 tumor (500mm ³) the mice loading dose of receiving treatment is 214mg/kg antibody, gives the antibody of 60mg/kg maintenance dose after 72 hours again.One week of the injection of antibodies first time (168 hours), the back was not (before observing tumour regression; See Figure 18 A) in the mice body, gather in the crops tumor and phosphorylation detection lysis buffer (seeing foregoing), carry out the homogenizing processing.With lysate 14000rpm centrifugal twice and measure protein concentration in the collected supernatant (Bio-Rad protein assay, Bio-Rad, Hercules, CA).The lysate (4mg) of each duplicate samples is carried out immunoprecipitation with anti--PDGFR Alpha antibodies.Make people PDGFR α behind the immunoprecipitation and anti-PDGFR Alpha antibodies or anti-phosphotyrosine antibody form immunoblotting then.Figure 19 is illustrated in these tumors, compare with the human IgG matched group to resist-the PDGFR Alpha antibodies causes the reduction of PDGFR α phosphotyrosine level.

[0172] cell line engineering.At first, the clones coding people anti--gene and the order-checking of PDGFR Alpha antibodies heavy chain and variable region of light chain.From MEDAREX obtain can with the primer series of 5 ' and 3 ' flanking sequence annealed combination of human normal immunoglobulin's variable region sequences the hybridoma that derives from MEDAREX.Primer is to AB88 (forward) and AB90 (oppositely) variable region of heavy chain (table 7) that is used to increase.The primer that comprises forward primer AB182 and reverse primer AB16 is to (table 7) the light chain product that can be used for increasing.With the 0.4kb product cloning of these reactions in carrier ZeroBlunt (Invitrogen) to produce AB88-1 (V _H) and AB 182-3 (V κ), institute's insertion sequence can use universal primer T7 and M13R to check order.

[0173] in order to obtain the plasmid vector of The expressed IgGl antibody, the variable region of having cloned by pcr amplification also connects by two steps and to form the expression vector that comprises the constant region gene.Use the masterplate of the plasmid AB88-1 of 25ng during first pcr amplification heavy chain as primer I PHF5 (forward) and IPHR5 (oppositely).Use the first set reaction liquid of 5 μ l as masterplate and primer OPSIF and IPHR5 during secondary PCR amplification heavy chain.Two forward primers are united use and can be contained 19 amino acid whose Mus heavy chain gene signal sequence (MGWSCIILFLVATATGVHS at coding; 5 ' end of immunoglobulin gene SEQ ID NO:24) increases by 57 base pairs, and immunoglobulin can be effectively handled and secrete.In addition, forward primer OPSIF can increase a total ＂ Kozak ＂ sequence (J MoI.Biol.196:947), make these genes in mammalian cell, can effectively be translated, can also increase by 5 ' HindIII restriction endonuclease sites clone and be amplified product to appropriate carriers.The heavy chain reverse primer comprises Nhel site in the structure, is used to be cloned into the constant region carrier.

[0174] use the Expand Buffer system #3 among the Expand PCR kit (Boehringer Mannheim Inc.) in the reaction system of 50 μ l, to carry out PCR according to operation instruction according to following cycling condition:

Once circulate 94 ° 2 minutes

Five times the circulation 94 ° 20 seconds

48 ° 60 seconds

68 ° 2 minutes

20 times the circulation 94 ° 20 seconds

65 ° 60 seconds

68 ° 2 minutes

1 time the circulation 68 ° 5 minutes

Through the row agarose gel electrophoresis of going forward side by side of purified product after the two-wheeled PCR, to carrier pDFc (figure .8), this carrier contains people γ 1 constant region with the postdigestive fragment cloning of HindIII-Nhel.

[0175] the pcr amplification light chain uses the pAB182-3 plasmid of 25ng as masterplate primer I PLF4 (forward) and IPLR2 (oppositely) for the first time.The pcr amplification light chain uses the primary reaction liquid of 5 μ l as masterplate and primer OPSIF and IPLR2 for the second time.For heavy chain, two forward primers provide secretory signal sequence.The light chain reverse primer comprises that the interior BsiWI site of a structure is used for the clone and enters κ constant region carrier pLck (figure .8).Carry out the PCR reaction according to above-mentioned heavy chain amplification method.Through after the two-wheeled PCR, the purified product row agarose gel electrophoresis of going forward side by side, to pLck, this carrier contains the human kappa light chain constant region with product cloning.

[0176] for obtain can stable transfection single plasmid vector, the heavy chain expression box that will contain CVM promoter, heavy chain coding region and polyA element is cloned into the light chain carrier as Notl-Sall digestion fragment (Figure 20).

[0177] this construct be used in the myeloma cell line NSO cell produce can steady production cell line.Carry out electroporation and use expression plasmid transfection NSO cell with BioRad Gene Pulser II.Before transfection, with PvuI with the plasmid DNA linearisation, solution (the 100ul dH that ethanol precipitation and resuspended one-tenth concentration are 0.4mg/ml ₂Contain 40ug among the O).Use 250 volt of 400 μ Fd pulse with cell and 40ugDNA electroporation in the final volume of 800ul.Cell behind the electroporation is dispersed in calf serum (the dFCS) (Hyclone that contains 10% process dialysis, Lot#:AHA7675) and in the 50ul aliquot in the DEME culture medium of 2mM glutamine (Invitrogen/LifeTechnologies) (JRH Biosciences Inc.), move in the hole of about 18 96 hollow plates, every hole density is 5,000-10,000 cell.Begin to screen glutamine synthetase (GS) positive transformant by adding the DEME that does not contain glutamine but contain 10% calf serum (dFCS) and be supplemented with Ix GS supplement (JRH Biosciences Inc.) after 24 hours.Cell is cultivated 2-4 week, 5% CO down at 37 ℃ ₂Can make colony growth and expansion.Screened more than 300 clone with Anti-Human Fc (γ) ELISA (horseradish peroxidase detects in the A450nm place).Clone's (58%) of amplification expressing antibodies also detects through the productivity after the cultivation in 3-5 days.Make cell adapted serum-free medium by the serum-free GS-OS culture medium amplification positive cell line that when going down to posterity, adds equal volume at every turn.Amplification can be produced 25ug/ml or more strong positive cell is used for further analyzing making it adapt to serum-free medium fully through three days inferior fusions after cultivate in (sub-confluent) 24 holes.

[0178] be to be understood that and predict be this professional field technical staff can production the present invention in the variant of disclosed any principle, and these are revised and all belong to category of the present invention.

Sequence table

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<400>62

<210>63

<211>233

<212>PRT

＜213〉people (Homo sapiens)

<400>63

Claims (80)

1. method for the treatment of the bone tumor experimenter, described method comprises the IGF-IR antagonist that gives effective dose.

2. one kind is suppressed the method that bone tumor is grown, and described method comprises the IGF-IR antagonist that gives effective dose.

3. method for the treatment of the bone tumor experimenter, described method comprises the PDGFR alpha-2 antagonists that gives effective dose.

4. one kind is suppressed the method that bone tumor is grown, and described method comprises the PDGFR alpha-2 antagonists that gives effective dose.

5. each method in the claim 1 to 4, wherein bone tumor is a primary tumor.

6. each method in the claim 1 to 4, wherein bone tumor is a secondary tumors.

7. each method in the claim 1 to 4, wherein growth of tumour cell is an androgen dependent form.

8. each method in the claim 1 to 4, wherein growth of tumour cell is non-androgen dependent form.

9. each method in the claim 1 to 4, wherein neoplasm metastasis is from carcinoma of prostate.

10. each method in the claim 1 to 4, wherein neoplasm metastasis is from breast carcinoma.

11. each method in the claim 1 to 4, wherein neoplasm metastasis is from pulmonary carcinoma.

12. the method for claim 1 or 2, wherein the IGF-IR antagonist is antibody or antibody fragment.

13. the method for claim 12, wherein said antibody or antibody fragment combine IGF-IR with comprising variable region of heavy chain with SEQIDNO:41 and the antibody competition with variable region of light chain of SEQ IDNO:51.

14. the method for claim 1 or 2, wherein the IGF-IR antagonist is the interior inhibitor of born of the same parents of IGF-IR.

15. the method for claim 14, wherein the IGF-IR inhibitor in the born of the same parents is selected from AG1024, NVP-AEW541 and BMS-554417.

16. the method for claim 3 or 4, wherein the PDGFR alpha-2 antagonists is antibody or antibody fragment.

17. the method for claim 16, wherein said antibody or antibody fragment combine PDGFR α with comprising variable region of heavy chain with SEQIDNO:8 and the antibody competition with variable region of light chain of SEQ ID NO:16.

18. the method for claim 3 or 4, wherein the PDGFR alpha-2 antagonists is the interior inhibitor of born of the same parents of PDGFR α.

19. the method for claim 18, wherein the PDGFR alpha inhibitor in the born of the same parents is selected from AG1296, STI-571 and SU11248.

20. the method for claim 12 or 16, antibody wherein or antibody fragment are the humanizeds.

21. the method for claim 12 or 16, antibody wherein or antibody fragment are humanized.

22. the method for claim 12 or 16, antibody wherein or antibody fragment are chimeric.

23. the method for claim 1 or 2, wherein the IGF-IR antagonist can suppress combining of IGF-I or IGF-II and IGF-IR.

24. the method for claim 1 or 2, wherein the IGF-IR antagonist can in and IGF-I or IGF-II to the activation of IGF-IR.

25. the method for claim 1 or 2, wherein the IGF-IR antagonist can reduce the concentration of IGF-IR surface receptor.

26. the method for claim 1 or 2, wherein the IGF-IR antagonist be can with K _dValue is about 3 x 10 ^-10M ^-1Or the bonded antibody of littler IGF-IR.

27. the method for claim 1 or 2, wherein the IGF-IR antagonist can suppress the phosphorylation of IGF-IR downstream signal transduction molecule.

28. the method for claim 3 or 4, wherein the PDGFR alpha-2 antagonists can suppress the somatomedin in platelet source and combining of PDGFR α.

29. the method for claim 3 or 4, wherein the PDGFR alpha-2 antagonists can in and the activation of PDGFR α.

30. the method for claim 3 or 4, wherein the PDGFR alpha-2 antagonists can reduce the concentration of PDGFR α surface receptor.

31. the method for claim 3 or 4, wherein the PDGFR alpha-2 antagonists is to be about 10 with the Kd value ^-9M ^-1Or the bonded antibody of littler IGF-IR.

32. the method for claim 3 or 4, wherein the PDGFR alpha-2 antagonists can suppress the phosphorylation of PDGFR α downstream signal transduction molecule.

33. the method for claim 3 or 4, wherein the PDGFR alpha-2 antagonists can suppress the inductive Akt activation of bone marrow.

34. each method among the claim 1-4 also comprises uniting and uses second kind of receptor tyrosine kinase antagonist.

35. the method for claim 1 or 2, wherein the IGF-IR antagonist is a bi-specific antibody.

36. the method for claim 35, wherein said bi-specific antibody has specificity to IGF-IR and PDGFR α.

37. the method for claim 35, wherein said bi-specific antibody has specificity to IGF-IR and EGFR.

38. the method for claim 3 or 4, wherein the PDGFR alpha-2 antagonists is a bi-specific antibody.

39. the method for claim 38, wherein said bi-specific antibody has specificity to PDGFR α and EGFR.

40. each method among the claim 1-4 also comprises the antitumor drug that gives effective dose.

41. the process of claim 1 wherein that described antitumor drug is a docetaxel.

42. the process of claim 1 wherein that described antitumor drug is an amycin.

43. the process of claim 1 wherein that described antitumor drug is lonizing radiation.

A 44. separation and purification and PDGFR α is had specific humanized's antibody or antibody fragment of coming comprises one or more and be selected from following complementary determining region: the SEQ IDNO:2 that is positioned at CDRH1; Be positioned at the SEQ ID NO:4 of CDRH2; Be positioned at the SEQ ID NO:6 of CDRH3; Be positioned at the SEQ ID NO:10 of CDRL1; Be positioned at the SEQ ID NO:12 and the SEQ ID NO:14 that is positioned at CDRL3 of CDRL2.

45. the antibody of claim 44 or antibody fragment comprise the SEQ IDNO:2 that is positioned at CDRH1, the SEQ ID NO:4 that is positioned at CDRH2 and the SEQ ID NO:6 that is positioned at CDRH3.

46. the antibody of claim 44 or antibody fragment comprise SEQ ID NO:8.

47. the antibody of claim 44 or antibody fragment comprise the SEQ ID NO:10 that is positioned at CDRL1, are positioned at the SEQ ID NO:12 of CDRL2 and are positioned at the SEQ ID NO:14 of CDRL3.

48. the antibody of claim 44 or antibody fragment comprise SEQ ID NO:16.

49. the antibody of claim 44 or antibody fragment comprise the SEQ IDNO:2 that is positioned at CDRH1, the SEQ ID NO:4 that is positioned at CDRH2, the SEQ ID NO:6 that is positioned at CDRH3, the SEQ ID NO:10 that is positioned at CDRL1, are positioned at the SEQ ID NO:12 of CDRL2 and are positioned at the SEQ ID NO:14 of CDRL3.

50. the antibody of claim 44 or antibody fragment comprise SEQ ID NO:8 and SEQ IDNO:16.

51. each antibody or antibody fragment among the claim 44-50 can combine with PDGFR α by selectivity.

52. each antibody or antibody fragment among the claim 44-50 can suppress combining of PDGFR α and PDGFR alpha ligands.

53. each antibody or antibody fragment among the claim 44-50, can in and PDGFR α.

54. each antibody or antibody fragment among the claim 44-50 are selected from: single-chain antibody, Fab fragment, strand Fv, double antibody and three antibody.

55. the conjugate of each antibody or antibody fragment among the claim 44-50.

56. the conjugate of claim 55 comprises antitumor drug, target part or acceptor portion.

57. the encoding antibody that a separation and purification obtains or the polynucleotide of antibody fragment comprise that one or more are selected from following nucleotide sequence: be positioned at CDRH1 SEQ ID NO:1, be positioned at CDRH2 SEQ ID NO:3, be positioned at CDRH3 SEQ ID NO:5, be positioned at CDRL1 SEQ ID NO:9, be positioned at the SEQ ID NO:11 of CDRL2 and be positioned at the SEQIDNO:13 of CDRL3.

58. the polynucleotide of separation and purification in the claim 57 comprise SEQ ID NO:7.

59. the polynucleotide of separation and purification in the claim 57 comprise SEQ ID NO:15.

60. an expression vector comprises among the claim 57-59 each polynucleotide.

61. a recombinant host cell comprises the expression vector of claim 60.

62. the recombinant host cell of claim 61 can produce polypeptide that comprises SEQ ID NO:8 and the polypeptide that comprises SEQ ID NO:16.

63. the recombinant host cell of claim 61 can produce the polypeptide that comprises SEQ IDNO:8 and SEQID NO:16.

64. the activated method of PDGFR α in the neutralization mammal comprises among the claim 44-54 that gives effective dose each described antibody.

65. a method that suppresses mammal tumor growth comprises among the claim 44-54 that treats effective dose each described antibody.

66. the method for claim 65, wherein said tumor PDGF-B expression R α.

67. the method for claim 65, wherein said tumor overexpression PDGFR α.

68. the method for claim 65, wherein said tumor are primary tumor.

69. the method for claim 65, wherein said tumor are metastatic tumo(u)r.

70. the method for claim 65, wherein said tumor are the intractable tumor.

71. the method for claim 65, wherein said tumor are the tumor that has formed blood vessel.

72. the method for claim 65, wherein said tumor is selected from: ovarian tumor, breast tumor, lung tumor, hepatocarcinoma, gastrointestinal stromal tumor, melanoma, renal cell carcinoma, tumor of prostate and soft tissue sarcoma.

73. the method for claim 65, wherein antibody or antibody fragment and antitumor drug coupling.

74. the method for claim 73, wherein antitumor drug is a chemotherapeutics.

75. the method for claim 73, wherein antitumor drug is an amycin.

76. the method for claim 73, wherein antitumor drug is lonizing radiation.

77. the method for claim 65, wherein antibody or antibody fragment and second kind of PDGFR alpha-2 antagonists are united use.

78. the method for claim 77, wherein the PDGFR alpha-2 antagonists is a PDGFR alpha-2 antagonists in the born of the same parents.

79. the method for claim 65 also comprises epithelial growth factor receptor (EGFR) antagonist for the treatment of effective dose.

80. the method for claim 65 also comprises IGF-1 (IGF-IR) antagonist for the treatment of effective dose.

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